L-dopa microbiome therapy

ABSTRACT

The present invention generally provides methods and compositions for the treatment of Parkinson’s disease and depression and/or anxiety. The invention relates to recombinant microorganisms, particularly gut-colonizing probiotics, modified to produce L-DOPA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. Serial No. 16/287,437, filed onFeb. 27, 2019, which claims priority to provisional applications U.S.Serial No. 62/785,980, filed Dec. 28, 2018, and U.S. Serial No.62/635,983, filed Feb. 27, 2018, which are incorporated herein byreference in their entireties.

SEQUENCE LISTING XML

The instant application contains a sequence listing, which has beensubmitted in XML file format by electronic submission and is herebyincorporated by reference in its entirety. Said XML file, created onDec. 9, 2022, is named P12428US03.xml and is 25,694 bytes in size.

FIELD OF THE INVENTION

This invention relates generally to a recombinant microbial cell,specifically to a probiotic strain engineered to produce L-DOPA, andmethods of using the same to provide L-DOPA in a sustained manner fortreatment of Parkinson’s disease and other Parkinsonian disorders. Usesof the recombinant microbial cell for treatment of depression, anxietyand other related mood disorders are also disclosed.

BACKGROUND OF THE INVENTION

More than 10 million people worldwide, and one million Americans haveParkinson’s disease (PD), and approximately 50,000 new cases arediagnosed each year, with the incidence among aging population exceedingthat for other younger segments of the US population. In addition,Parkinsonian disorders including progressive supranuclear palsy (PSP)and multiple system atrophy (MSA) have overlapping neurological deficitsand clinical pathology with PD. The cardinal pathology of PD isprogressive neurodegeneration of dopamine-producing neurons insubstantia nigra, contributing to dopamine deficiency that manifest insevere motor symptoms including rigidity, bradykinesia, tremors, andpostural instability. The amino acid L-DOPA is the precursor to theneurotransmitter dopamine, and has been used for the treatment of avariety of neurological disorders including PD. The discovery ofdopamine replacement therapy with Levodopa (L-DOPA) for PD representsone of the most remarkable success stories in the history of medicine.For decades, L-DOPA is the drug most often prescribed because it’sunparalleled symptomatic relief to PD patients. Unfortunately, L-DOPAgold standard therapy met inherent side effects commonly referred toL-DOPA induced dyskinesia (LID). Although neurochemical basis of LID isnot completely understood, dopamine receptor sensitization due topulsated delivery of L-DOPA in form of 100-500 mg tablet 2 times day isconsidered to be major reason for the LID. The clinical diagnosis ofL-DOPA drug fluctuations include peak dose, off period dystonia, anddiphasic dyskinesia. In the face of antiparkinsonian treatment, the lackof effective treatment to control LID remains by far the mostchallenging problem.

Therefore, it is an object of the present invention to provide methodsand compositions to deliver L-DOPA in a sustained manner therebyavoiding pulsated delivery and the L-DOPA induced dyskinesia associatedwith the standard treatment regimen. Other objects will become apparentfrom the description of the invention which follows.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and compositions comprising arecombinant microbial cell capable of producing L-DOPA, for use in thetreatment of Parkinson’s disease and depression and/or anxiety. In someembodiments, the recombinant microbial cell colonizes the gut of thesubject in need of treatment, thereby providing L-DOPA in a sustainedmanner.

In one embodiment, the disclosure provides a method for providing asubject in need thereof with a treatment for Parkinson’s diseasecomprising administering to the subject an effective amount of acomposition comprising a recombinant microbial cell capable of producingL-DOPA.

In another embodiment, methods of treating depression and/or anxiety andimproving motivation to do difficult tasks are provided. The methodcomprises administering to the subject in need thereof an effectiveamount of a composition comprising a recombinant microbial cell capableof producing L-DOPA.

In the preceding embodiments of the invention, the recombinant microbialcell can be a probiotic. In an exemplary embodiment, the probiotic is E.coli Nissle 1917. In some embodiments, the composition is administeredorally. In some embodiments, the composition is administered onalternate days. In certain embodiments, the subject is a mammal. In apreferred embodiment the mammal is a human. In some embodiments, theeffective amount of the recombinant microbial cell comprises from about10⁶ CFU to about 10¹² CFU. In a preferred embodiment, the effectiveamount comprises about 10⁹ CFU of the recombinant microbial cell.

In some embodiments, an effective amount of a DOPA decarboxylaseinhibitor is co-administered with the recombinant microbial cellcomposition. In a preferred embodiment, the DOPA decarboxylase inhibitoris carbidopa or benserazide. In certain embodiments, the compositionfurther comprises L-Tyrosine. In other embodiments, supplementalL-Tyrosine is not required.

In some embodiments, the recombinant microbial cell capable of producingL-DOPA comprises a hpaB nucleotide sequence and a hpaC nucleotidesequence. In some embodiments, the hpaB nucleotide sequence is thesequence set forth in SEQ ID NO: 1 and the hpaC nucleotide sequence isthe sequence set forth in SEQ ID NO: 2. In another embodiment, therecombinant microbial cell capable of producing L-DOPA comprises thehpaBC_(syn) nucleotide sequence set forth in SEQ ID NO: 3.

In yet another embodiment, a pharmaceutical composition for use in thetreatment of Parkinson’s disease is provided. In some embodiments, thepharmaceutical composition is for use in the treatment of depressionand/or anxiety.

In some embodiments, the pharmaceutical composition comprises arecombinant microbial cell capable of producing L-DOPA and colonizingthe gut of a subject, thereby providing L-DOPA in a sustained manner. Insome embodiments, recombinant microbial cell of the pharmaceuticalcomposition is a probiotic. In an exemplary embodiment, the probiotic isE. coli Nissle 1917.

In some embodiments, the pharmaceutical composition comprises from about10⁶ CFU to about 10¹² CFU of the recombinant microbial cell. In apreferred embodiment, the pharmaceutical composition comprises about 10⁹CFU of the recombinant microbial cell. In some embodiments, thepharmaceutical composition further comprises L-Tyrosine. In anotherembodiment, the pharmaceutical composition further comprises apharmaceutically acceptable carrier.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the figures anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1 depicts the biosynthesis of L-DOPA by E.coli4-hydroxyphenylacetate 3-monooxygenase (HpaB) and its FAD reductase(HpaC).

FIG. 2A shows a schematic experimental setup. FIG. 2B shows HPLC profileand L-DOPA standard curve. Top: HPLC peak profile shows a distinctL-DOPA peak in the recombinant E.coli bacterial media identical toL-DOPA standards. Bottom: L-DOPA standard curve. FIG. 2C shows thequantification of L-DOPA yield. L-DOPA-producing E. coli DH10B weregrown in 10 ml LB medium containing kanamycin (50 µg/ml), L-tyrosine (1mg/ml), and ascorbic acid (1-10 mg/ml) for 0-24 h at 37° C. At each timepoint (0, 3, 6, 9, 12 and 24 h), cultured cell suspension were obtainedand cell-free broth was collected for HPLC analysis. FIG. 2D shows theimprovement of L-DOPA yield by allowing 12-h recovery growth formbacterial glycerol stock in LB media before inoculation into 10 mML-tyrosine-containing LB media. FIG. 2E shows L-DOPA production inNissle 1917 strain. L-DOPA-producing E. coli Nissle 1917 were grown in10 ml LB medium containing kanamycin (50 µg/ml), L-tyrosine (1-10mg/ml), and ascorbic acid (1-10 mg/ml) for 0-12 h at 37° C. L-DOPAproduction was determined by HPLC.

FIGS. 3A-3D show that MitoPark Parkinson’s disease mice displayprogressive motor deficits, dopaminergic neuronal loss, and depletion ofstriatal dopamine. FIG. 3A shows moving track, FIG. 3B shows rotarodperformance, FIG. 3C shows TH immunocytochemistry (DAB stain), and FIG.3D shows depletion of striatal dopamine.

FIGS. 4A-4D show that MitoPark mice display progressive spatial learningdeficits and olfactory dysfunction. FIGS. 4A-4B show Morris water mazetest, and FIGS. 4C-4D show social discrimination test.

FIGS. 5A-5F show gut dysfunction in MitoPark mice. FIG. 5A shows wholegut transit time. FIG. 5B shows colon transit time. FIG. 5C shows rateof colonic motility. FIG. 5D shows colon length. Representativephotographs showing smaller colon length in 24-wk old MitoPark micecompared to control mice are shown on the right. FIG. 5E shows dopaminelevels in the colon of 24-wk old MitoPark mice compared to control mice.FIG. 5F shows representative 60× IHC Images showing decreased number ofTH positive neurons in the myenteric plexus of the colon in MitoParkmice.

FIGS. 6A-6B show C57 black mice that orally received hpaBC E. coli orPBS daily for 7 days. FIG. 6A shows colonies counts. Fecal pellets werecollected 6 and 24 h post treatment and subjected to kanamycin-selectivecolonies count. FIG. 6B shows L-DOPA levels. Two colonies were pickedand grown in LB medium containing kanamycin (50 µg/ml), L-tyrosine (10mg/ml), and ascorbic acid (1-10 mg/ml) for 12 h. L-DOPA levels in mediumwere assessed by HPLC. HpaBC E. coli was used as a positive control.

FIGS. 7A-7D show C57 black mice that orally received hpaBC E. coli orPBS daily for 7 days. Before sacrifice, mice were tested for locomotoractivities. FIG. 7A shows moving track. FIG. 7B shows horizontalactivity. FIG. 7C shows total distance. FIG. 7D shows striatal tissuescollected and assayed for dopamine levels.

FIG. 8 shows E. coli Nissle 1917 L-DOPA-producing bacteria (EcN¹_(L)-_(DOPA)) successfully colonizes the gut of PD mouse model. Fifteento seventeen week old C57BL/6 mice received single oral administrationof EcN¹ _(L-DOPA) and were co-treated with the decarboxylase inhibitorbenserazide. Fecal pellets were collected and kanamycin-resistantcolonies were counted and plotted as CFU/g of fecal pellet.

FIGS. 9A-9B show the pharmacokinetic profile of E. coli Nissle 1917L-DOPA-producing bacteria (EcN¹ _(L-DOPA)) in mouse model of PD. C57BL/6mice received single oral administration of EcN¹ _(L-DOPA) and wereco-treated with the decarboxylase inhibitor benserazide. FIG. 9A showstime course of Plasma L-DOPA levels and FIG. 9B shows plasma L-DOPAlevels post 24 hr treatment. Plasma was collected prior to treatment andon days 1, 2 and 10 post-treatment and stored frozen. Plasma L-DOPAlevels were then quantified using the HPLC electrochemical detectionmethod.

FIGS. 10A-10D show E. coli Nissle 1917 L-DOPA-producing bacteria (EcN¹_(L-) _(DOPA)) alleviates Parkinsonian motor symptoms in the chronic,progressively degenerative MitoPark mouse model of PD. Sixteen-week-oldMitoPark (MP) and Littermate control (LM) mice (male and female, n=3)received oral administration of 10⁹ CFU/150 ul of either EcN¹ _(L-DOP)or EcN_(Vec) containing L-Tyrosine (100 mg/kg) and were co-treated withbenserazide (12.5 mg/kg i.p.) on alternate days. Animals were assessedweekly for 4 weeks for exploratory locomotor activity via an open-fieldtest recorded using the Versamax computerized activity monitoringsystem. Representative data shown for age 20 weeks. FIG. 10A showsVersamax Motor activity map, FIG. 10B shows horizontal activity, FIG.10C shows total distance travelled, and FIG. 10D shows stereotypicactivity.

FIG. 11 shows E. coli Nissle 1917 L-DOPA-producing bacteria (EcN¹_(L)-_(DOPA)) reduce anxiety-like behavior in Control mice. Heat map ofanimal’s exploratory behavior in Versamax open-field test. Quadrant 2(Q2) is the most openly exposed quadrant. Animals were assessed weeklyfor 4 weeks for anxiety using the Versamax and forced swim test.Representative data shown for age 20 weeks. See brief description ofFIGS. 10 for animal treatment paradigm.

FIGS. 12A-12B show E. coli Nissle 1917 L-DOPA-producing bacteria (EcN¹_(L-) _(DOPA)) reduce depression-like behavior in Control and MitoParkmice in Forced Swim Test. FIG. 12A shows time immobile and FIG. 12Bshows distance travelled in forced swim test. Animals were assessedweekly for 4 weeks for depression-like behavior. Representative datashown for age 20 weeks. See brief description of FIGS. 10 for animaltreatment paradigm.

FIG. 13 shows E. coli Nissle 1917 L-DOPA-producing bacteria (EcN¹_(L)-_(DOPA)) protect against progressive loss of vestibulomotorfunction and motor coordination. Animals were assessed weekly forvestibulomotor function and motor coordination using the Rotarod systemevery week for 4 weeks. Representative data shown for age 20 weeks. Seebrief description of FIGS. 10 for animal treatment paradigm.

FIG. 14A shows the re-construction of L-DOPA-expressing plasmids. FIG.14B shows quantification of L-DOPA yield. The newly generatedL-DOPA-expressing systems produced significantly higher amounts ofL-DOPA compared to the RSF1030-based EcN¹ _(L-DOPA) system, even withoutadding L-tyrosine. ***, p<0.001, compared to EcN¹ _(L-) _(DOPA), byone-way ANOVA, n=4-6.

FIGS. 15A-15B show C57 black mice orally received a single dose of EcN²_(L-DOPA) (10⁹ CFU) or PBS, both in combination with benserazide (Bz).FIG. 15A shows colonization profile. Fecal pellets were collected dailyfor 4 days and subjected to qPCR-based analysis of colonization. FIG.15B shows plasma L-DOPA levels in EcN² _(L-DOPA)-treated mice asdetermined by HPLC. Data represented as mean ± SEM from 6 mice.

FIGS. 16A-16D show C57 black mice orally received a single dose of EcN²_(L-DOPA) (10⁹ CFU) or PBS, in combination with a single oral dose of Bz(50 mg/kg, 3 times/day) for 1 and 2 days. FIG. 16A shows HPLC detectionof plasma Bz at 30 h after the first Bz treatment. FIGS. 16B-16C showsL-DOPA levels in plasma and striatal tissues analyzed by HPLC. FIG. 16Dshows qPCR analysis of colonization from intestinal content scraped offfrom various gut segments. (***, p<0.01 by one-way ANOVA). n=6.

FIGS. 17A-17B show C57 black mice treated with a single daily dose ofEcN¹ _(L-) _(DOPA) (10⁹ CFU) in combination with carbidopa (10 mg/kg,i.p.) for 1 week. FIG. 17A shows representative H&E stained ileum, cecumand colon sections from EcN¹L-_(DOPA)-treated mice. n=3. FIG. 17B showstaxonomy bar chart. Genomic DNA isolated from fecal pellets weresubjected to PCR amplication of V4 variable region of 16S rRNA performedat the ISU DNA facility on the Illumina MiSeq Platform. The sequenceswere analyzed using QIIME. The taxonomic summary at the family level wasshown. xyz, unnamed family. n=4.

FIGS. 18A-18C show MitoPark (MP) and age-matched control mice (11-16weeks old, male and female, n=4-5) received oral administration of 10⁹CFU of either of EcN² _(L-DOPA) or PBS and co-administered with a singledose of Bz (12.5 mg/kg i.p.) on alternate days for 1 week. Animals wereassessed weekly for exploratory locomotor activity via an open-fieldtest recorded using the Versamax computerized activity monitoringsystem. Representative data shown for post 1-week treatment. FIG. 18Ashows Versamax Motor activity map, FIG. 18B shows horizontal activity,and FIG. 18C shows stereotypic activity. (*, p<0.05 by one-way ANOVA).ns, not significant.

FIG. 19 shows C57 black mice received oral administration of 10⁹ CFU ofeither of EcN_(Vec) or PBS and co-administered with a single dose of Bz(12.5 mg/kg i.p.) for 1 day. Plasma L-DOPA levels were analyzed by HPLC.n=3.

FIG. 20 shows plasmid constructs used for expression of hpaBC in EcN.Synthetic hpaBC genes were cloned into the pRHAM vector yielding theplasmid shown. The rha promoter and operator were subsequently replacedwith 3 individual constitutive promoters (P1, P2, P3). Along with thecoding regions for hpaB and hpaC, additional plasmid features include:kan, kanamycin resistance; rop, control of plasmid copy number;ColEIori, origin of replication (SEQ ID NOs: 4-23).

FIGS. 21A-21C show C57 black mice orally received a single dose of EcN²_(L-D)o_(PA) (10⁹ CFU) or PBS, in combination with a single oral dose ofBz (12.5 mg/kg, ip) for 2-16 h. (A-C) NE, 5-HT and DA levels in frontalcortex tissues were analyzed by HPLC. (***, p<0.01 by one-way ANOVA).n=3-6. FIG. 21A shows norepinephrine levels, FIG. 21B shows5-hydroxytryptamine levels, and FIG. 21C shows dopamine levels.

FIGS. 22A-22C shows mice (15-17 weeks old, n=3-8) received oraladministration of 10⁹ CFU of either of EcN² _(L-D)o_(PA) or PBS andco-administered with a single dose of Bz (12.5 mg/kg i.p.) on alternatedays. Animals were assessed weekly for 4 weeks for anxiety-like (FIGS.22A-22B) and depression-like behavior (FIG. 22C). FIG. 22A shows EcN²_(L-DOPA) reduces anxiety-like behavior in C57 mice. Left:Representative heat map of animal’s exploratory behavior in VersaMaxOFT. Quadrants (Q3,4) are the open exposed quadrants while Q1,2 areclosed. FIG. 22B shows elevated plus maze (EPM). Right: quantitation ofhorizontal activities. Representative data shown for post 3-weektreatment. Center: time spent in open arms. Right: total distance inEPM. Representative data shown for post 4-week treatment. FIG. 22C showsforced swim test (FST). Top: Move tracks are shown. Bottom left: timeimmobile. Bottom right: distance travelled in FST. Representative datashown for post 2-week treatment. (*, p<0.05, **, p<0.01, ***, p<0.001 byStudent’s t-test).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions comprising arecombinant microbial cell capable of producing L-DOPA. The recombinantmicrobial cell colonizes the gut of the subject in need of treatment andprovides L-DOPA in a sustained manner to avoid the development ofLevodopa-induced dyskinesia (LID).

So that the present invention may be more readily understood, certainterms are first defined. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which embodiments ofthe invention pertain. Many methods and materials similar, modified, orequivalent to those described herein can be used in the practice of theembodiments of the present invention without undue experimentation, thepreferred materials and methods are described herein. In describing andclaiming the embodiments of the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The singular terms “a”, “an”, and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicate otherwise.The word “or” means any one member of a particular list and alsoincludes any combination of members of that list.

Numeric ranges recited within the specification, including ranges of“greater than,” “at least”, or “less than” a numeric value, areinclusive of the numbers defining the range and include each integerwithin the defined range. For example, when a range of “1 to 5” isrecited, the recited range should be construed as including ranges “1 to4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like.

The term “about” as used herein, refers to variation in the numericalquantity that can occur, for example, through typical measuringtechniques and equipment, with respect to any quantifiable variable,including, but not limited to, mass, volume, time, distance, wavelength, frequency, voltage, current, and electromagnetic field. Further,given solid and liquid handling procedures used in the real world, thereis certain inadvertent error and variation that is likely throughdifferences in the manufacture, source, or purity of the ingredientsused to make the compositions or carry out the methods and the like. Theterm “about” also encompasses amounts that differ due to differentequilibrium conditions for a composition resulting from a particularinitial mixture. The term “about” also encompasses these variations.Whether or not modified by the term “about,” the claims includeequivalents to the quantities.

As used herein, the terms “microbe”, “microbial cell”, or“microorganism” refer to an organism of microscopic, submicroscopic, orultramicroscopic size that typically consists of a single cell. Examplesof microorganisms include bacteria, viruses, fungi, certain algae, andprotozoa. The term “microbial” indicates pertaining to, orcharacteristic of a microorganism.

The term “microbiome”, as used herein, refers to a population ofmicroorganisms from a particular environment, including the environmentof the body or a part of the body. The term is interchangeably used toaddress the population of microorganisms itself (sometimes referred toas the microbiota), as well as the collective genomes of themicroorganisms that reside in the particular environment. The term“environment,” as used herein, refers to all surrounding circumstances,conditions, or influences to which a population of microorganisms isexposed. The term is intended to include environments in a subject, suchas a human and/or animal subject.

The recombinant cell according to the invention may be constructed fromany suitable host cell. The host cell may be an unmodified cell or mayalready be genetically modified. In one embodiment, the cell is arecombinant microbial cell. In one embodiment, the recombinant microbialcell is recombinant gut-colonizing microbial cell. In one embodiment,the cell can be a prokaryotic cell or a eukaryotic cell. In oneembodiment, the cell is a prokaryotic cell.

In one embodiment, the recombinant microbial cell is a nonpathogenicbacterial cell. In some embodiments, the recombinant microbial cell is acommensal bacterial cell. In some embodiments, the recombinant microbialcell is a yeast cell. In some embodiments, the recombinant microbialcell is a probiotic. In some embodiments, the recombinant microbial cellis a naturally pathogenic microbial cell that is modified or mutated toreduce or eliminate pathogenicity.

“Probiotic” is used to refer to live, non-pathogenic microorganisms,e.g., bacteria, which can confer health benefits to a host organism thatcontains an appropriate amount of the microorganism. Some species,strains, and/or subtypes of non-pathogenic microorganisms are currentlyrecognized as probiotic. Examples of probiotic microorganisms include,but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus,and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium,Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillusbulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, andSaccharomyces boulardii (Dinleyici et al., 2014; U.S. Pat. Nos.5,589,168; 6,203,797; 6,835,376).

Examples of probiotic bacteria include, but are not limited to, specificprobiotic strains of Lactobacillus, Bifidobacterium, Lactococcus,Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, orEscherichia coli.

In some embodiments, a probiotic Lactobacillus may include, withoutlimitation, a Lactobacillus reuteri, Lactobacillus plantarum,Lactobacillus casei (such as Lactobacillus casei Shirota), Lactobacillussalivarius, Lactobacillus paracasei, Lactobacillus lactis, Lactobacillusacidophilus, Lactobacillus sakei, Lactobacillus brevis, Lactobacillusbuchneri, Lactobacillus fermentum, Lactobacillus delbrueckii,Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus,Lactobacillus garvieae, Lactobacillus acetotolerans, Lactobacillusagilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillusamylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus,Lactobacillus animalis, Lactobacillus aviarus, Lactobacillusbifermentans, Lactobacillus bulgaricus, Lactobacillus carnis,Lactobacillus caternaformis, Lactobacillus cellobiosis, Lactobacilluscollinoides, Lactobacillus confuses, Lactobacillus coryniformis,Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillusdivergens, Lactobacillus farciminis, Lactobacillus fructivorans,Lactobacillus fructosus, Lactobacillus gallinarum, Lactobacillusgasseri, Lactobacillus graminis, Lactobacillus haiotoierans,Lactobacillus hamster, Lactobacillus heterohiochii, Lactobacillushilgardii, Lactobacillus homohiochii, Lactobacillus iners, Lactobacillusintestinalis, Lactobacillus jensenii, Lactobacillus johnsonii,Lactobacillus kandleri, Lactobacillus kefiri, Lactobacilluskefuranofaciens, Lactobacillus kefirgranum, Lactobacillus kunkeei,Lactobacillus leichmannii, Lactobacillus llndnerl, Lactobacillusmalefermentans, Lactobacillus mall, Lactobacillus maltaromicus,Lactobacillus manihotivorans, Lactobacillus minor, Lactobacillusminutus, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillusnagelii, Lactobacillus oris, Lactobacillus panis, Lactobacillusparabuchneri, Lactobacillus paracasei, Lactobacillus parakefiri,Lactobacillus paralimentarius, Lactobacillus paraplantarum,Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus piscicola,Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus rhamnosus,Lactobacillus rhamnosus GG, Lactobacillus rimae, Lactobacillus rogosae,Lactobacillus ruminis, Lactobacillus sanfranciscensis, Lactobacillussharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillusuli, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillusxylosus, Lactobacillus yamanashiensis, or a Lactobacillus zeae.

In some embodiments, a probiotic Escherichia coli may be E. coli Nissle1917. As used herein, the term “Escherichia” refers to a genus ofGram-negative, non-spore forming, facultatively anaerobic, rod-shapedbacteria from the family Enterobacteriaceae. The genusEscherichiainclude various species, such asEscherichia coli. The terms “Escherichiacoli Nissle 1917” or “EcN” as used herein refer to a non-pathogenicGram-negative probiotic bacteriaEscherichia coli strain that is capableof colonizing the human gut. In an exemplary embodiment, the probioticis theEscherichia coli strain Nissle 1917.

Escherichia coli Nissle 1917 has evolved into one of the bestcharacterized probiotics (Ukena et al., 2007). The strain ischaracterized by its complete harmlessness (Schultz, 2008), and has GRAS(generally recognized as safe) status (Reister et al., 2014). E. colistrain Nissle 1917 lacks defined virulence factors such asalpha-hemolysin, other toxins, and mannose-resistant hemagglutinatingadhesins (Blum et al. Infection. 23(4):234-236 (1996)), P-fimbrialadhesins, and the semirough lipopolysaccharide phenotype and expressesfitness factors such as microcins, ferritins, six different iron uptakesystems, adhesins, and proteases, which support its survival andsuccessful colonization of the human gut (Grozdanov et al. J Bacteriol.186(16): 5432-5441 (2004)). As early as in 1917, E. coli Nissle waspackaged into medicinal capsules, called MUTAFLOR®, for therapeutic use.E. coli Nissle has since been used to treat ulcerative colitis in humansin vivo (Rembacken et al., 1999), to treat inflammatory bowel disease,Crohn’s disease, and pouchitis in humans in vivo (Schultz, 2008), and toinhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella invitro (Altenhoefer et al., 2004). It is commonly accepted that E. coliNissle’s therapeutic efficacy and safety have convincingly been proven(Ukena et al., 2007).

Examples ofEscherichia coli Nissle 1917 bacteria include those availableas DSM 6601 from the German Collection for Microorganisms inBraunschweig, Germany or commercially as the active component inMUTAFLOR® (Ardeypharm GmbH, Herdecke, Germany).

In some embodiments, a probiotic Bifidobacterium may be Bifidobacteriuminfantis, Bifidobacterium adolescentis, Bifidobacterium animalis subspanimalis, Bifidobacterium longum, Bifidobacterium fidobacterium breve,Bifidobacterium bifidum, Bifidobacterium animalis subsp, lactis orBifidobacterium lactis, such as Bifidobacterium lactis DN-173 010.

In some embodiments, a probiotic Bacillus may be Bacillus coagulans. Insome embodiments, a probiotic Lactococcus may be Lactococcus lactissubsp. Lactis such as Lactococcus lactis subsp. lactis CV56. In someembodiments, a probiotic Enterococcus may be Enterococcus durans. Insome embodiments, a probiotic Streptococcus may be Streptococcusthermophilus.

In some embodiments, the probiotic bacterium may be an auxotrophicstrain designed, for example, to limit its survival outside of the humanor animal intestine, using standard techniques.

The probiotic may be a variant or a mutant strain of bacterium (Arthuret al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayredeet al., 2006). Non-pathogenic bacteria may be genetically engineered toenhance or improve desired biological properties, e.g., survivability.Non-pathogenic bacteria may be genetically engineered to provideprobiotic properties. Probiotic cells may be genetically engineered toenhance or improve probiotic properties, e.g., enhance gut colonization.

Bacterial strains can be readily obtained using standard methods knownin the art. For example, a commensal bacterium such asEscherichia coliNissle 1917 can be obtained from a commercial preparation of theprobiotic MUTAFLOR®. Bacteria can be cultured using standard methodsknown in the art.

One of ordinary skill in the art would appreciate that the geneticmodifications disclosed herein may be modified and adapted for otherspecies, strains, and subtypes of bacteria or other microorganisms.

“Gut” refers to the organs, glands, tracts, and systems that areresponsible for the transfer and digestion of food, absorption ofnutrients, and excretion of waste. In humans, the gut comprises thegastrointestinal tract, which starts at the mouth and ends at the anus,and additionally comprises the esophagus, stomach, small intestine, andlarge intestine. The gut also comprises accessory organs and glands,such as the spleen, liver, gallbladder, and pancreas. The uppergastrointestinal tract comprises the esophagus, stomach, and duodenum ofthe small intestine. The lower gastrointestinal tract comprises theremainder of the small intestine, i.e., the jejunum and ileum, and allof the large intestine, i.e., the cecum, colon, rectum, and anal canal.Bacteria can be found throughout the gut, e.g., in the gastrointestinaltract, and particularly in the intestines.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, the termincludes reference to the specified sequence as well as thecomplementary sequence thereof.

As used herein, the terms “peptide”, “polypeptide”, and “protein” willbe used interchangeably to refer to a chain of amino acids each of whichis joined to the next amino acid by a peptide bond. In one aspect, thisterm also includes post translational modifications of the polypeptide,for example, glycosylations, acetylations, phosphorylations and thelike. Included within the definition are, for example, peptidescontaining one or more analogues of an amino acid or labeled amino acidsand peptidomimetics.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogs of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

In one aspect, there is provided an isolated nucleic acid moleculecomprising 4-hydroxyphenylacetate 3-monooxygenase (HpaB) and its FADreductase (HpaC). In one embodiment, the nucleic acid molecule has atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3. In one embodiment,the nucleic acid molecule comprises or consists of the nucleic acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3. In oneembodiment, the nucleic acid molecule is a vector. In one embodiment,the nucleic acid molecule is a vector comprising an insert comprising orconsisting of SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3. In oneembodiment, transformation of a microbial cell with the vector resultsin the production of L-DOPA. Additionally, other HpaB and HpaC nucleicacid sequences may be identified through databases such as Genbank.

In one aspect, the present disclosure is directed towards recombinantEscherichia coli Nissle 1917(EcN) cell, or a variant thereof,transformed with a nucleic acid molecule containing one or more genesinvolved in the biosynthesis of L-DOPA. As demonstrated in the Examples,the inventors have determined that EcN cells transformed with HpaB andHpaC are useful for the recombinant production of L-DOPA.

Accordingly, in one embodiment there is provided a recombinant EcN cellor variant thereof comprising HpaB (SEQ ID NO: 1) and HpaC (SEQ ID NO:2), or HpaBC (SEQ ID NO: 3). Optionally, the recombinant EcN cellcomprises one or more genes with sequence identity to HpaB (SEQ ID NO:1), HpaC (SEQ ID NO: 2), or HpaBC (SEQ ID NO: 3). For example, in oneembodiment, the cell comprises one or more nucleic acid sequences withat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQID NO: 3.

Sequence identity can be determined according to sequence alignmentmethods known in the art. Examples of these methods includecomputational methods such as those that make use of the BLASTalgorithm, available online from the National Center for BiotechnologyInformation. Sequence identity is most preferably assessed by thealgorithm of BLAST version 2.1 advanced search. BLAST is a series ofprograms that are available, for example, online from the NationalInstitutes of Health. References to BLAST searches are: Altschul, S. F.,Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic localalignment search tool.” J. Mol. Biol. 215:403410; Gish, W. & States, D.J. (1993) “Identification of protein coding regions by databasesimilarity search.” Nature Genet. 3:266272; Madden, T. L., Tatusov, R.L. & Zhang, J. (1996) “Applications of network BLAST server” Meth.Enzymol. 266:131_141; Altschul, S. F., Madden, T. L., Schaffer, A. A.,Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLASTand PSI_BLAST: a new generation of protein database search programs.”Nucleic Acids Res. 25:33893402; Zhang, J. & Madden, T. L. (1997)“PowerBLAST: A new network BLAST application for interactive orautomated sequence analysis and annotation.” Genome Res. 7:649656.

Percent sequence identity or homology between two sequences isdetermined by comparing a position in the first sequence with acorresponding position in the second sequence. When the comparedpositions are occupied by the same nucleotide or amino acid, as the casemay be, the two sequences are conserved at that position. The degree ofconservation between two sequences is often expressed as a percentagerepresenting the ratio of the number of matching positions in the twosequences to the total number of positions compared. Two nucleic acid,or two polypeptide sequences are substantially homologous to each otherwhen the sequences exhibit at least about 70%-75%, preferably 80%-82%,more preferably 85%-90%, even more preferably 92%, still more preferably95%, and most preferably 98% sequence identity over a defined length ofthe molecules, as determined using the methods above.

Nucleic acid hybridization may also be used to identify substantiallysimilar nucleic acid molecules to those reported herein. The presentnucleic acid molecules described herein may be used to identify genesencoding substantially similar polypeptides/proteins expected to havesimilar function. Nucleic acid hybridization may be conducted understringent conditions. Substantially similar sequences are defined bytheir ability to hybridize, under the following stringent conditions(0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS, 65° C.).

“Regulatory elements” refer to nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory elements may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences. Regulatory elements present on a recombinant DNAconstruct that is introduced into a cell can be endogenous to the cell,or they can be heterologous with respect to the cell. The terms“regulatory element” and “regulatory sequence” are used interchangeablyherein.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. An“inducible” or “repressible” promoter is a promoter which is underenvironmental control. Examples of environmental conditions that mayaffect transcription by inducible promoters include anaerobic conditionsor the presence of light. Tissue specific, tissue preferred, cell typespecific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterwhich is active under most environmental conditions. In someembodiments, the promoter comprises one or more of SEQ ID NOs: 4-23. Inan exemplary embodiment, the promoter sequence comprises SEQ ID NO: 6.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

As used herein, “vector” refers to a DNA or RNA molecule (such as aplasmid, linear piece of DNA, cosmid, bacteriophage, yeast artificialchromosome, or virus, among others) that carries nucleic acid sequencesinto a host cell. The vector or a portion of it can be inserted into thegenome of the host cell.

As used herein the term “codon-optimized” refers to the modification ofcodons in the gene or coding regions of a nucleic acid molecule toreflect the typical codon usage of the host organism without alteringthe polypeptide encoded by the nucleic acid molecule. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of the host organism. A “codon-optimized sequence” refersto a sequence, which was modified from an existing coding sequence, ordesigned, for example, to improve translation in an expression host cellor organism of a transcript RNA molecule transcribed from the codingsequence, or to improve transcription of a coding sequence. Codonoptimization includes, but is not limited to, processes includingselecting codons for the coding sequence to suit the codon preference ofthe expression host organism. Many organisms display a bias orpreference for use of particular codons to code for insertion of aparticular amino acid in a growing polypeptide chain. Codon preferenceor codon bias, differences in codon usage between organisms, is allowedby the degeneracy of the genetic code, and is well documented among manyorganisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

As used herein, “transformation” refers to a process of introducing anexogenous nucleic acid molecule (e.g, a vector, a recombinant DNAmolecule) into a host cell. Transformation typically achieves a geneticmodification of the cell. The introduced nucleic acid may integrate intoa chromosome of a cell, or may replicate autonomously. A cell that hasundergone transformation, or a descendant of such a cell, is“transformed” and is a “recombinant” cell. Recombinant cells aremodified cells as described herein. Cells herein may be transformedwith, for example, one or more of a vector, a plasmid or a linear piece(eg., a linear piece of DNA created by linearizing a vector) of DNA. Theplasmid or linear piece of DNA may or may not comprise a selectable orscreenable marker. In an exemplary embodiment, the recombinant cellcomprises the hpaBC genes for the biosynthesis of L-DOPA fromL-tyrosine.

A nucleic acid may be introduced into a cell by conventional methods,such as, for example, electroporation (see, e.g., Heiser W. C.Transcription Factor Protocols: Methods in Molecular Biology™ 2000; 130:117-134), chemical (e.g., calcium phosphate or lipid) transfection (see,e.g., Lewis W. H., et al., Somatic Cell Genet. 1980 May; 6(3): 333-47;Chen C., et al., Mol Cell Biol. 1987 August; 7(8): 2745-2752), fusionwith bacterial protoplasts containing recombinant plasmids (see, e.g.,Schaffner W. Proc Natl Acad Sci USA. 1980 April; 77(4): 2163-7),transduction, conjugation, or microinjection of purified DNA directlyinto the nucleus of the cell (see, e.g., Capecchi M. R. Cell. 1980November; 22(2 Pt 2): 479-88).

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

In one embodiment, a method for providing a subject with a treatment forParkinson’s disease is provided. In another embodiment, methods oftreating depression or anxiety and methods of improving motivation to dodifficult tasks are provided. The method comprises administering to thesubject in need thereof an effective amount of a composition comprisinga recombinant microbial cell of the invention.

Methods of treating other disorders associated with dopamine are alsocontemplated. The most common disease characterized by a dopamineproduction deficiency is Parkinson’s disease; however, invention may bereadily adapted for the treatment of other diseases characterized byinsufficiency of dopamine production. In one embodiment of theinvention, a method of treating a disorder resulting fromdopamine-related dysfunction is provided.

In one embodiment, the method of the invention is intended for treating,preventing, managing and/or delaying the progression of Parkinson’sdisease, restless leg syndrome, depression, stress, obesity, chronicposttraumatic stress disorder, anxiety disorders, obsessive-compulsivedisorders, postpartum depression; schizophrenia, narcolepsy, manic,bipolar, and affective disorder; executive function disorders, such asattention deficit disorder (ADHD), Tourette syndrome and autism;cocaine, amphetamine, alcohol dependency, and addictive behavior, suchas pathological gambling. The diseases and conditions enumerated aboveare given by way of example and not by way of limitation.

As used herein, the term “treating” means ameliorating, improving orremedying a disease, disorder, or symptom of a disease or condition. Forexample, with respect to Parkinson’s disease, treatment may be measuredby quantitatively or qualitatively to determine the presence/absence ofthe disease, or its progression or regression using, for example,symptoms associated with the disease or clinical indications associatedwith the pathology.

As used herein, the term “subject”, “individual”, or “patient” refers toany organism upon which embodiments of the invention may be used oradministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. In some embodiments, a subject is a mammal, e.g.,a human or non-human primate (e.g., an ape, monkey, orangutan, orchimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle,or cow.

As used herein a “pharmaceutical composition” refers to a preparation ofrecombinant microbial cells of the invention with other components suchas a pharmaceutically acceptable carrier and/or excipient.

As used herein, the term “pharmaceutically acceptable carrier” refers toany carrier, diluent, excipient, wetting agent, buffering agent,suspending agent, lubricating agent, adjuvant, vehicle, delivery system,emulsifier, disintegrant, absorbent, preservative, surfactant, colorant,flavorant, or sweetener, preferably non-toxic, that would be suitablefor use in a pharmaceutical composition. The compositions of the presentinvention may be administered in the form of a pharmaceuticalcomposition with a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers may be chosen to permit oral administration oradministration by any other known route.

The term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples include, but are not limited to, calciumbicarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils, polyethylene glycols,and surfactants, including, for example, polysorbate 20.

Pharmaceutical compositions comprising a recombinant microbial cell canbe formulated to be suitable for oral administration, for example asdiscrete dosage forms, such as, but not limited to, tablets (includingwithout limitation scored or coated tablets), pills, caplets, capsules,chewable tablets, powder packets, cachets, troches, wafers, aerosolsprays, or liquids, such as but not limited to, syrups, elixirs,solutions or suspensions in an aqueous liquid, a non-aqueous liquid, anoil-in-water emulsion, or a water-in-oil emulsion. Such compositionscontain a predetermined amount of the pharmaceutically acceptable saltof the disclosed compounds, and may be prepared by methods of pharmacywell known to those skilled in the art. See generally, Remington: TheScience and Practice of Pharmacy, 21st Ed., Lippincott, Williams, andWilkins, Philadelphia, Pa. (2005).

In addition to the oral dosing, noted above, the compositions of thepresent invention may be administered by any suitable route, in the formof a pharmaceutical composition adapted to such a route, and in a doseeffective for the treatment intended. The compositions may, for example,be administered parenterally, e.g., intravascularly, intraperitoneally,subcutaneously, or intramuscularly. For parenteral administration,saline solution, dextrose solution, or water may be used as a suitablecarrier. In an embodiment of the invention, the therapeutic compositioncontaining the recombinant microbial cells may be administeredintrarectally. A rectal administration preferably takes place in theform of a suppository, enema, or foam.

As used herein, the terms “pharmaceutically effective” or“therapeutically effective” shall mean an amount of a composition thatis sufficient to show a meaningful patient benefit, i.e., treatment,prevention, amelioration, or a decrease in the frequency of thecondition or symptom being treated.

The compositions and methods described herein can be administered to asubject in need of treatment, e.g. in need of treatment for Parkinson’sdisease, depression, or anxiety. In some embodiments, the methodsdescribed herein comprise administering an effective amount ofcompositions described herein, e.g. recombinant microbial cells to asubject in order to alleviate a symptom. As used herein, “alleviating asymptom” is ameliorating any condition or symptom associated with agiven condition. As compared with an equivalent untreated control, suchreduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%or more as measured by any standard technique.

In certain embodiments, an effective dose of a composition comprisingrecombinant microbial cells as described herein can be administered to apatient once. In certain embodiments, an effective dose of a compositioncomprising recombinant microbial cells can be administered to a patientrepeatedly. In some embodiments, the dose can be a daily administration,for example oral administration, of, e.g., a capsule comprising cells asdescribed herein.

In some embodiments, the effective amount of the recombinant microbialcell is from about 10⁶ CFU to about 10¹³ CFU. Therefore, in someembodiments, the effective amount of the recombinant microbial cell isabout 10⁶, about 10⁷, about 10⁸, about 10⁹, about 10¹⁰, about 10¹¹,about 10¹², or about 10¹³ CFU. In some preferred embodiments, theeffective amount is about 10⁹ CFU of the recombinant microbial cell. Insome embodiments, the effective amount results peak plasma levelssimilar to that of the standard tablet form of L-DOPA treatment. In someembodiments, the effective amount achieves stable therapeutic plasmaL-DOPA concentrations of from about 300 to about 1600 ng/ml over timewith the recombinant microbial cell as compared to traditional L-DOPA.Therefore, in some embodiments, the effective amount effective amountachieves stable therapeutic plasma L-DOPA concentrations of about 300ng/ml, about 400 ng/ml, about 500 ng/ml, about 600 ng/ml, about 700ng/ml, about 800 ng/ml, about 900 ng/ml, about 1000 ng/ml, about 1100ng/ml, about 1200 ng/ml, about 1300 ng/ml, about 1400 ng/ml, about 1500ng/ml, about 1600 ng/ml, or more. In some preferred embodiments, theeffective amount results in peak plasma levels reaching about 1500ng/ml. The optimal dose of the recombinant microbial cell maximizes gutcolonization without inducing toxicity, including gut tissue damage,inflammation or gut microbial dysbiosis.

A composition comprising recombinant microbial cells can be administeredover a period of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period. The administration can be repeated, forexample, on a regular basis, such as hourly for 3 hours, 6 hours, 12hours, daily (i.e. one a day), every other day (i.e. on alternate days),or longer or such as once a week, or biweekly (i.e., every two weeks)for one month, two months, three months, four months or longer.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing, schedulecan vary from once a week to daily depending on a number of clinicalfactors, such as the subject’s sensitivity to recombinant microbialcells.

The desired dose or amount of activation can be administered at one timeor divided into subdoses, e.g., 2-4 subdoses and administered over aperiod of time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more.

The dosage ranges for the administration of recombinant microbial cells,according to the methods described herein depend upon, for example, theform of the cells, their potency, and the extent to which symptoms,markers, or indicators of a condition described herein are desired to bereduced, for example the percentage reduction desired. The dosage shouldnot be so large as to cause adverse side effects. Generally, the dosagewill vary with the age, condition, and sex of the patient and can bedetermined by one of skill in the art. The dosage can also be adjustedby the individual physician in the event of any complication.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. A composition may beformulated such that a unit dose of the composition contains a specifiednumber of microorganisms.

The efficacy of recombinant microbial cells in, e.g. the treatment of acondition described herein can be determined by the skilled clinician.However, a treatment is considered “effective treatment,” as the term isused herein, if any one or all of the signs or symptoms of a conditiondescribed herein are altered in a beneficial manner, other clinicallyaccepted symptoms are improved, or even ameliorated, or a desiredresponse is induced following treatment according to the methodsdescribed herein. Efficacy can be assessed, for example, by measuring amarker, indicator, symptom, and/or the incidence of a condition treatedaccording to the methods described herein or any other measurableparameter appropriate. Efficacy can also be measured by a failure of anindividual to worsen as assessed by hospitalization, or need for medicalinterventions (i.e., progression of the disease is halted). Methods ofmeasuring these indicators are known to those of skill in the art and/orare described herein. Treatment includes any treatment of a disease inan individual or an animal (some non-limiting examples include a humanor an animal) and includes: (1) inhibiting the disease, e.g., preventinga worsening of symptoms; or (2) relieving the disease, e.g., causingregression of symptoms. An effective amount for the treatment of adisease means that amount which, when administered to a subject in needthereof, is sufficient to result in effective treatment as that term isdefined herein, for that disease. Efficacy of an agent can be determinedby assessing physical indicators of a condition or desired response. Itis well within the ability of one skilled in the art to monitor efficacyof administration and/or treatment by measuring any one of suchparameters, or any combination of parameters. Efficacy can be assessedin animal models of a condition described herein. When using anexperimental animal model, efficacy of treatment is evidenced when astatistically significant change in a marker is observed.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

As used herein, the term “combination therapy” refers to theadministration of the recombinant microbial cell with an at least oneadditional pharmaceutical or medicinal agent (e.g., an anxiolyticagent), either sequentially or simultaneously.

The methods described herein can further comprise administering a secondagent and/or treatment to the subject, e.g. as part of a combinatorialtherapy. In certain embodiments of the present invention, therecombinant microbial cells can be used in combination therapy with atleast one other therapeutic agent.

In certain embodiments, the pharmaceutical compositions, and methods forthe treatment further comprise one or more therapeutic agents fortreating Parkinson’s disease selected from a dopaminergic agent, such asLevodopa-carbidopa (SINEMET®, SINEMET CR®) or Levodopa-benserazide(PROLOPA®, MADOPAR®, MADOPAR HBS®); a dopaminergic and anti-cholinergicagent, such as amantadine (SYMMETRYL®, SYMADINE®); an anti-cholinergicagent, such as trihexyphenidyl (ARTANE®), benztropine (COGENTIN®),ethoproprazine (PARSITAN®), or procyclidine (KEMADRIN®); a dopamineagonist, such as apomorphine, bromocriptine (PARLODEL®), cabergoline(DOSTINEX®), lisuride (DOPERGINE®), pergolide (PERMAX®), pramipexole(MIRAPEX®), or ropinirole (REQUIP®); a MAO-B (monoamine oxidase B)inhibitor, such as selegiline or deprenyl (ATAPRYL®, CARBEX®,ELDEPRYL®); a COMT (catechol O-methyltransferase) inhibitor, such asCGP-28014, tolcapone (TASMAR®) or entacapone (COMTAN®); or othertherapeutic agents, such as baclofen (LIORESAL®), domperidone(MOTILIUM®), fludrocortisone (FLORINEF®), midodrine (AMATINE®),oxybutynin (DITROPAN®), propranolol (INDERAL®, INDERAL-LA®), clonazepam(RIVOTRIL®), or yohimbine.

The other therapeutic agent can be an anti-depression agent. Usefulanti-depression agents include, but are not limited to, amitriptyline,clomipramine, doxepine, imipramine, triripramine, amoxapine,desipramine, maprotiline, nortriptyline, protripyline, fluoxetine,fluvoxamine, paroxetine, setraline, venlafaxine, bupropion, nefazodone,trazodone, phenelzine, tranylcypromine and selegiline.

The other therapeutic agent can be an anxiolytic agent. Usefulanxiolytic agents include, but are not limited to, benzodiazepines, suchas alprazolam, chlordiazepoxide, clonazepam, clorazepate, diazepam,halazepam, lorazepam, oxazepam, and prazepam; non-benzodiazepine agents,such as buspirone; and tranquilizers, such as barbituates.

Levodopa (L-DOPA), an aromatic amino acid, is a white, crystallinecompound, slightly soluble in water, with a molecular weight of 197.2.It is designated chemically as(-)-L-a-amino-b-(3,4-dihydroxybenzene)propanoic acid. Its empiricalformula is C₉H₁₁NO₄, and its structural formula is

Current evidence indicates that symptoms of Parkinson’s disease arerelated to depletion of dopamine in the corpus striatum. Administrationof dopamine is ineffective in the treatment of Parkinson’s diseaseapparently because it does not cross the blood-brain barrier. L-DOPA isable to cross the protective blood-brain barrier and enter the brain,where it is further converted into dopamine by the enzyme DOPAdecarboxylase (DDC). Because L-DOPA can be converted into dopaminewithin the peripheral nervous systems, which may contribute toL-DOPA-related adverse side effects, L-DOPA is conventionally given incombination with a peripheral DDC inhibitor, such as carbidopa orbenserazide to prevent its breakdown in the bloodstream, so more L-DOPAcan enter the brain.

In some embodiments, the methods of the invention compriseco-administering a DOPA decarboxylase inhibitor. In certain embodiments,decarboxylase enzyme inhibitor is carbidopa, a carbidopa prodrug,benserazide, methylphenidate, or a combination thereof.

All publications, patents and patent applications identified herein areincorporated by reference, as though set forth herein in full. Theinvention being thus described, it will be apparent to those skilled inthe art that the same may be varied in many ways without departing fromthe spirit and scope of the invention. Such variations are includedwithin the scope of the following claims.

The invention is further illustrated by the following specific exampleswhich are not intended in any way to limit the scope of the invention.

EXAMPLES Example 1: Cloning and Characterization of L-DOPA ProducingBacteria

Several different L-DOPA biosynthesis pathways have been characterizedin an attempt to build and maintain a parallel metabolism in bacteria.The commonly used fungal or plant tyrosinase pathway has previouslyproven effective at oxidizing L-tyrosine to L-DOPA is prone to secondaryoxidation of L-DOPA to dopaquinone. Another well-characterized L-DOPAbiosynthesis pathway relies on tyrosine hydroxylase. While this pathwaysuffers from less overoxidation, it requires a complex regenerationpathway for the essential pterin cofactor.

As an alternative to these two more well characterized pathways, weinvestigated the biosynthesis of L-DOPA from L-tyrosine using theplasmid-based expression of E. coli hpaBC genes (FIG. 1 ). First, webuilt and validated several different L-DOPA production plasmids usingthe 15A, RSF1030 and broad host range RSF1010 origins of replication,and a range of common constitutive and inducible promoters (P_(EM7),P_(CP25) and P_(lacUV5)). These plasmids have been shown to produceL-DOPA, as determined by oxidation to easily observed black DOPApolymers, in several different E. coli hosts (DH10B, BL21 and OP50), aswell as in Serratia species.

Example 2: Characterization and Quantification of Microbial L-DOPASynthesis via a Plasmid-Based Expression System

We quantitatively assayed L-DOPA production using an internallystandardized high performance liquid chromatography (HPLC) method. Forthis, a time-course study was performed to determine the optimalincubation time for the production of L-DOPA. A schematic representationof the experimental setup is shown in FIG. 2A. Five microliter ofdefrosted glycerol stock of E. coli (strain DH10B) containing the L-DOPAproducing plasmid RSF 1030 were grown in 10 ml Luria-Bertani (LB) mediumcontaining kanamycin (50 µg/ml), L-tyrosine (1 mg/ml), and ascorbic acid(1 mg/ml) present to prevent oxidation of L-DOPA for 0-24 h at 37° C. Ateach time point (0, 3, 6, 9, 12 and 24 h), small aliquots (0.5 ml) ofcultured cell suspension were obtained and cell-free broth was thencollected by centrifuge/filtration. HPLC analysis of the cell-free brothwas carried out on a C18 column as previously described. As depicted inFIG. 2B, the HPLC peak profile of the 24-h grown cell-free broth showeda distinct peak at the retention time 3.01 min, which corresponded toL-DOPA standards, confirming the production of L-DOPA. Calculation ofthe L-DOPA yield from a L-DOPA standard curve (FIG. 2B, bottom panel)indicated that L-DOPA production started after 3 h incubation with ayield of 110 ng/ml, stayed constant over the next 9 h, but significantlyincreased to 350 ng/ml at the 24 h (FIG. 2C, left panel). A cellsuspension without L-tyrosine was also included as a negative control,which showed no L-DOPA production. Furthermore, increasing theL-tyrosine substrate concentration to 10 mM dramatically enhanced L-DOPAproduction, with the highest yield of L-DOPA (528 ng/ml) achieved at 12h incubation (FIG. 2C, right panel). HPLC data will be confirmed andcorrelated with small molecule mass spectrometry data. To furtherpotentiate analysis of L-DOPA production, we recovered the E. colistrain from the -80° C. glycerol stock before inoculation intoL-tyrosine/ascorbic acid-containing LB media. Five microliter of E. coliglycerol stock was recovered to grow for 12 h in 5-mL LB medium, and 5µl of cell suspension was then subcultured for 12 h in 5-ml LB mediumsupplemented with kanamycin, ascorbic acid and 10 mg/ml L-tyrosine. HPLCanalysis revealed that performing a recovery step significantly enhancedL-DOPA production (FIG. 2D).

Example 3: Transformation of hpaBC Gene Into E. Coli Nissle 1917 Strainfor L-DOPA Production

Since the non-pathogenic probiotic E. coli strain Nissle 1917 is asuccessful colonizer of the human gut and has shown therapeutic utilityfor treatment of various intestinal disorders (Grozdanov, Raasch et al.2004), we transformed the L-DOPA-producing plasmid RSF 1030 into Nissle1917 strain and tested its production of L-DOPA. As shown in FIG. 2E,growing this hpaBC Nissle 1917 strain in LB medium containing L-tyrosine(1-10 mg/ml) results in a rapid and similar production of L-DOPA.Collectively, these data indicate our E. coli DH10B and Nissle 1917strains presents an effective source for L-DOPA production.

Example 4: C. Elegans Model of PD

As a first test of whether microbes could generate L-DOPA in a form thatwould be usable by organisms, we used the L-DOPA-producing bacteria torescue a movement defect in a worm model of PD. The C. elegans cat-2mutant lacks the enzyme TH. Wild-type worms switch efficiently fromswimming to crawling forms of motion when exiting a puddle. However,although the cat-2 mutant worm swims and crawls normally, it becomestransiently paralyzed as it attempts to switch from a swimming to acrawling gait. As with many animals, dopamine mediates this locomotorytransition because treatment with exogenous dopamine rescues themovement defect (Vidal-Gadea et al., 2013). In our pilot studies, wefound that feeding the L-DOPA producing bacteria to the cat-2 mutantenables it to move like wild-type worms. Control cat-2 mutants fedstandard bacteria (OP50 strain) or standard bacteria supplemented withtyrosine showed feeble progress reflected in their tracks as they left apuddle at the center of a plate in comparison to WT. In contrast, cat-2mutants fed the L-DOPA-producing E. coli strain described above and thensupplemented with tyrosine displayed tracks qualitatively identical toWT. The rescue of the Parkisonian phenotype was also observed when wequantified the percent of individual worms that migrated away. Thesebehavioral results provide strong evidence that the bacteria producesufficient quantities of L-DOPA with specific exogenous bioactivity.

Example 5: MitoPark Mouse Model of PD

MitoPark mouse model was developed by using Cre/LoxP recombination toselectively knockout the mitochondrial transcription factor TFAM incells expressing DAT. We currently have a breeding colony established atIowa State University’s animal facility. This model has a chronic,spontaneous, and progressive neurodegeneration accompanied byprogressive motor deficits, intraneuronal inclusions, and dopamine loss.As shown in FIGS. 3A-B, MitoPark motor performance at 8 weeks of agedoes not differ from age-matched controls. However, at 12 weeks of age,these mice displayed significantly reduced locomotor activity, and thesedeficits became more severe at 24 weeks of age. As previously reported,the progressive motor dysfunction is accompanied by a progressive lossof dopaminergic neurons in the substantia nigra (FIG. 3C) and areduction of striatal dopamine (FIG. 3D). Importantly, this model isL-DOPA responsive and dyskinesia develops over time, making it an idealpre-clinical model to test the response of microbiome-generated L-DOPA.Furthermore, recent studies showed that non-motor aspects of the PD areinvolved in this model. We recently confirmed that MitoPark mice displaysome clinically relevant nonmotor symptoms of PD, such as olfactorydysfunction (FIGS. 4A-B) and spatial learning deficits (FIGS. 4C-D).Given that gastrointestinal (GI) disturbances are one of the earliestnon-motor symptoms affecting most patients with PD, we evaluated GIdysfunction in this model. Curiously, from 8 weeks of age, MitoPark miceshowed a lower whole gut transit time, indicating more GI motility inthese mice compared to aged-matched controls (FIG. 5A). Since themotility pattern may vary from one intestinal segment to another, wefurther evaluated regional motility by measuring colon transit time. Asshown in FIG. 5B, the MitoPark mice had increased colon transit timecompared to age-matched controls. Similarly, the rate of bead movementin the distal colon was significantly decreased in the MitoPark groupcompared to the control group (FIG. 5C). Interestingly, we observedsmaller colon lengths in MitoPark mice when compared to that of thecontrol mice (FIG. 5D). Next, since dopamine plays an important role inmodulating GI motility, we assessed intestinal dopamine content viaHPLC. Reduced dopamine levels were observed in 24-week old MitoPark micecompared to age matched controls (FIG. 5E), whereas the levels of3,4-Dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-HTwere unchanged in these mice compared to control mice. Along withdopaminergic neurodegeneration in the mid-brain, we also observed areduction in the number of dopaminergic neurons in the gut (FIG. 5F).All together, these preliminary data suggest the MitoPark mouse modelrecapitulates the chronology and development of GI dysfunction alongwith other motor and non-motor symptoms and can become an attractivemodel for pre-clinical assessment of the efficacy ofmicrobiome-generated L-DOPA.

Example 6: Characterization of Orally Administered L-DOPA Producing E.Coli Into the Gut of C57 Black Mice

We used the C57 black mice for testing whether our orally administeredL-DOPA-producing E. coli can colonize the gut of mice and effectivelyproduce L-DOPA. The treatment paradigm and experimental end-points aredepicted in Scheme 1.

Preparation of Fresh Inocula

L-DOPA-producing E. coli (strain: pRSF1030-hpaBC) were grown forovernight in 10 ml LB media containing kanamycin (100 µg/ml). After12-14 h, the OD₆₀₀ was measured and the colony forming units (CFU/ml)were calculated from the standard curve. Bacterial cells were thenpelleted and resuspended at 10⁸ CFU/150 µl in 1X PBS. The inocula isready to be administered to mice.

Oral Administration of L-DOPA Producing Bacteria Into MitoPark Mice

Nine 12-week-old C57 black mice were prescreened for baseline motoractivity and then randomized into 2 groups of 3 animals each. Firstgroup was received 150 µl 1X PBS daily for 7 days and sacrificed on day8; second group received of E. coli pRSF30-hpaBC (10⁸ CFU/150 µl 1X-PBS)daily for 7 days and sacrificed on day 8. Fecal pellets were collecteddaily 6 h and 24 h post-gavage. Animals were monitored for motoractivity using Versamax on day 7. On day 8, animals were sacrificed,blood was collected by cardiac puncture, and striatal and gut tissueswere collected and subjected to neurochemical and biochemicalassessments.

Assessment of Fecal Pellets for L-DOPA-Producing Bacteria

As expected, kanamycin selection on agar plates, where equal amount offecal pellet suspension from PBS- and hpaBC E. coli-treated groups wasspread, revealed that, in hpaBC E. coli-treated animals, 6 h time pointpellets contained a significantly higher number of kanamycin-positivebacterial colonies as compared to 24 h time point pellets (FIG. 6A).Only minimal kanamycin-positive colonies (less than 3) were observed inthe agar plates where fecal pellets from PBS control animals wereplated, suggesting that our E. coli pRSF30-hpaBC strain was successfullycolonized in the gut of mice.

Assessment of Kanamycin-Positive Bacterial Colonies for the Presence ofhpaBC Gene and L-DOPA Production

Next, we isolated 3 kanamycin-positive bacterial colonies from agarplates spread with 6 h and 24 h fecal pellet suspension of each mouseand then grew them in 10 ml of LB media containing kanamycin (100µg/ml), L-tyrosine (10 mg/ml), and ascorbic acid (1 mg/ml) for 12 h.

qPCR

5 ml of the cell medium were centrifuged, DNA was isolated frombacterial pellets and qPCR was performed using the primers specific forhpaBC gene.

As shown in FIG. 6B, kanamycin-positive colonies isolated from fecalpellets of hpaBC E. coli-treated group showed significant amplificationof the hpaBC gene, suggesting that the kanamycin-positive colonies fromhpaBC E. coli-treated animals indeed contain hpaBC gene coding for theenzyme that produces L-DOPA. The colonies picked from PBS controlanimals failed to grow in LB-kanamycin media. HPLC: 500 µl of LB-mediacontaining bacteria were centrifuged, then both media and bacterialpellets were processed for HPLC quantification of L-DOPA as describedabove. As shown in FIG. 6C, bacterial pellets and media from hpaBC E.coli-treated animals showed high amounts of L-DOPA production (range:98.34 - 161 ng/ml).

Behavioral Assessment

Behavioral analysis using Versamax computerized monitor activity systemrevealed that oral administration of L-DOPA-producing bacteria up to 7days did not have any adverse effects on moving paths, horizontalactivity and total distances (FIGS. 7A-C).

Plasma L-DOPA Levels

As shown in FIG. 7D, the plasma collected from hpaBC E. coli-treatedanimals contained significantly higher levels of L-DOPA as compared tothat from PBS control animals.

Striatal Dopamine Levels

Dopamine levels in the striatal tissues were increased by at least 50%in mice orally administered with L-DOPA-producing bacteria for 7 days ascompared to PBS administered animals, suggesting that microbiallydelivered L-DOPA is able to cross BBB and is further converted todopamine (FIG. 7E).

Serum Chemistry Profiling

Results revealed that orally administered E. coli pRSF30-hpaBC bacteriato C57 black mice did not alter serum chemistry profiling, suggestingthe safety of using this bacteria for therapeutic purposes (Table 1).

TABLE 1 Blood chemistry analysis results Treatment Paradigm PBS-treatedEc-LD-treated Albumin (Abaxis) 3 3.3 Alk Phos (Abaxis) 5 5 ALT (Abaxis)42 53 Amylase 587 591 T. Bilirubin (Abaxis) 0.25 0.2 BUN (Abaxis) 10 12Calcium (Abaxis) 4 4 Creatinine (Abaxis) 0.25 0.3 Glucose (Abaxis) 190.5190 Sodium (Abaxis) 151 148 Potassium - Abaxis 8.2 2.9 T. Protein(Abaxis) 3.5 3.4 Hemolytic Index (Ab) 2.5 3 Lipemic Index (Abaxis) 1 0Icteric Index (Abaxis) 0 0

Together, these results suggest the orally administered E. colipRSF30-hpaBC bacteria into the gut of C57 black mice successfullycolonize in the GI-tract. Further, L-DOPA that is produced in the gutcrosses the intestinal barrier and goes to the blood. Once in the blood,the L-DOPA travels to brain and gets converted to dopamine, resulting inincreased striatal dopamine levels. Behavioral assessment and serumchemistry analysis revealed that microbially delivered L-DOPA does notcause any change in behavior or serum chemistry, suggesting it isnon-toxic and safe.

Example 7: E. Coli Nissle 1917 L-DOPA-Producing Bacteria (EcN¹_(L-DOPA)) Successfully Colonize the Gut of Mouse Model

As described in FIGS. 2E, E. coli Nissle 1917 L-DOPA-producing bacteria(EcN¹ _(L-) _(DOPA)) were generated by transforming plasmid pRSF1030,which contains the hpaBC gene coding for L-DOPA synthesizing enzymes. Wealso generated an empty vector strain of E. coli Nissle 1917 bytransforming the pRSF plasmid/vector alone for use as a negativecontrol. This vector control will be referred to as EcN_(Vec).

Twelve- to 14-week-old male and female C57BL/6 mice (n=6) wereadministered a single oral gavage of 10⁶ or 10⁹ CFU/150 µl of EcN¹_(L-DOPA) containing L-Tyrosine (100 mg/kg) plus a single i.p. injectionof benserazide (12.5 mg/kg), an L-amino acid decarboxylase inhibitor.Fecal pellets were collected prior to treatment to establish baselinemeasurements and then daily for six days post-treatment. Fecal pelletswere homogenized with a bullet blender and plated in triplicates onto LBagar plates containing kanamycin (50 ug/ml). Kanamycin-resistantcolonies representing the EcN¹ _(L-DOPA) Nissle 1917 strain were countedand plotted as CFU/g of fecal pellet. Colonization of EcN¹ _(L-DOPA) atboth doses of 10⁶ and 10⁹ CFU/150 µl occurs in a time- anddose-dependent manner, peaking in 2-3 days, and then slowly decreasescloser to baseline levels over a period of six days (FIG. 8 ).Kanamycin-resistant colonies peaked 2 days after administering 10⁹CFU/150 µl EcN¹ _(L-DOPA) at 0.5×10⁶ CFU/g of fecal pellet, whereas itpeaked 3 days after administering 10⁶ CFU/150 µl EcN¹ _(L-DOPA) at0.18×10⁶ CFU/g of fecal pellet. Based on these results, we used EcN¹_(L-DOPA) at a dose of 10⁹ CFU/150 µl for subsequent efficacy studies.

Example 8: E. Coli Nissle 1917 L-DOPA Producing Bacteria (EcN¹_(L-DOPA)) Increase Plasma L-DOPA Levels to a Therapeutic Level in aMouse Model of PD

Approximately 15-17 week-old male and female C57BL/6 mice (n=6) receiveda single oral administration of 10⁹ CFU/150 µl of EcN¹ _(L-DOPA) orEcN_(Vec) containing L-tyrosine (100 mg/kg) plus a single i.p. injectionof benserazide (12.5 mg/kg). Plasma samples collected prior toadministration and again on days 1, 2 and 10 were subjected to HPLCanalysis for measurements of L-DOPA levels as previously described inour publications. Plasma L-DOPA levels peaked on day 2 after a singleoral administration of EcN¹ _(L-DOPA) and then declined over a period often days (FIG. 9A). Notably, the peak plasma level reached 1500 ng/ml,which is similar to the standard tablet form of L-DOPA treatment in PDpatients. In contrast, EcN_(Vec) administration did not increase plasmaL-DOPA levels as expected (FIG. 9B). The pharmacokinetic profile alsoparalleled the colonization profile (FIG. 8 ). Together the results fromthe colonization and PK profile reveal that alternate-day administrationof EcN¹ _(L-DOPA) will provide stable plasma levels of L-DOPA, which mayprovide great clinical benefits by avoiding the dose fluctuations anddyskinesia associated with current L-DOPA treatment. We adopted analternate-day EcN¹ _(L-DOPA) treatment paradigm for preclinical efficacystudies in the animal models described below.

Example 9: E. Coli Nissle 1917 L-DOPA-Producing Bacteria (EcN¹_(L-DOPA)) Alleviate Parkinsonian Neurological Symptoms, Depressive andAnxiety-Like Behavior in the Chronic, Progressively DegenerativeMitoPark Mouse Model of PD

MitoPark motor performance at age 8 weeks does not differ fromage-matched controls. However, starting at age 12 weeks, these micedisplay significantly reduced locomotor activity that progressivelyworsens with age. It is the best progressive degenerative mouse of PDavailable to date. At 16 weeks, MitoPark mice exhibit a PD phenotype. Wetreated 16-week-old male and female MitoPark and control mice(n=6/group) via oral administration of 10⁹ CFU/150 µl of EcN¹ _(L-DOPA)or EcN_(Vec) containing L-tyrosine (100 mg/kg) and i.p. injection ofbenserazide (12.5 mg/kg) on alternate days from 16-20 weeks. Wild-typelittermate mice from the MitoPark breeding colony were used as controlmice. Animals were assessed weekly for a) exploratory locomotor activityvia an open-field test (OFT) recorded using the Versamax computerizedactivity monitoring system, b) vestibulomotor function and motorcoordination using the Rotarod, and c) depression- and anxiety-likebehavior using the forced swim test (FST) and the Versamax OFT,respectively.

Attenuation of PD-Related Movement Deficits in the MitoPark TransgenicMouse Model

MitoPark mice at age 20 weeks displayed significantly reduced locomotoractivity compared to control mice (FIGS. 10A-D). Oral administration ofEcN¹ _(L-DOPA) increased locomotor activity in both MitoPark and controlmice as seen in the representative computerized motor activity plots(FIG. 10A). Quantitative analysis of the data embedded in the Versamaxmotor activity plot revealed that EcN_(vec)-administered 20-weekMitoPark mice exhibited decreased horizontal locomotor activity (FIG.10B), total distance traveled (FIG. 10C), stereotypy (FIG. 10D) comparedto EcN_(vec)-administered control mice; in the Versamax, stereotypyarises from grooming and head-bobbing, which are more common in micethat are not physically or behaviorally distressed. Oral administrationof EcN ¹ _(L-DOPA) significantly alleviated the deficits in horizontalactivity, total distance traveled and stereotypy in MitoPark mice withno significant effect in control mice. However, oral administration ofEcN_(Vec) did not have any significant effects on motor deficits ineither MitoPark or control mice (FIGS. 10A-D). Together this studysuggests that EcN¹ _(L)-_(DOPA) alleviates key neurological deficits inthe MitoPark mouse model of PD.

Anti-Depressive and Anti-Anxiety Effect of EcN¹ _(L-DOPA) in Mouse Model

Depression and anxiety assessments were performed using the FST and theVersamax OFT, respectively. To measure anxiety, the OFT exploits amouse’s natural tendency to hug walls, especially opague ones, and toavoid openly exposed areas when spontaneously exploring a novelenvironment. Thus, more time spent in zones or quadrants farthest fromopaque walls (i.e., most openly exposed) can serve as an index ofreduced anxiety. In this unforced locomotor assessment, Control micereceiving EcN¹ _(L-) _(DOPA) were significantly less anxious thanEcN_(vec)-treated mice (FIG. 11 ). The FST subjects mice to aninescapable, stress-inducing scenario that invariably leads to episodesof behavioral despair, or giving up, as measured by immobility. Variousmeasures of immobility, including latency, count, cumulative duration,and even distance, can be used to gauge motivational deficits linked todepression. To quantify immobility, we used ANY-maze computerizedvideo-tracking and analysis software, as described in our recentpublications, which employs a mobility threshold that allows for onlythose minor movements necessary to tread water. Thus, higher immobilityscores and shorter distances represent a more depressive phenotype,whereas lower immobility and longer distances reflect a more motivated,stress-coping phenotype. In FST, MitoPark mice at age 20 weeks weresignificantly more immobile than age-matched control mice, suggestingthat Parkinsonian mice express a more depressive phenotype than docontrol mice (FIG. 12A). However, MitoPark mice administered with EcN¹_(L-DOPA) were significantly less immobile compared toEcN_(Vec)-administered MitoPark mice. Importantly, when compared toEcN_(Vec)-administered control mice, control mice receiving EcN¹_(L-DOPA) were not only significantly less immobile, their increasedactivity exceeded the mobility threshold enough to show that theirbodies cumulatively moved a significantly greater distance. Thissuggests the mice were highly motivated to escape rather than just treadwater, further indicating that EcN¹ _(L-) _(DOPA) can be an effectivetreatment for depression and motivational impairments in otherwisehealthy individuals. Collectively, our FST and OFT results clearlydemonstrate the anti-depressive and anti-anxiety effects of EcN¹_(L-DOPA) in both control and PD mouse models.

Improvement in Vestibulomotor Function and Motor Coordination

This assessment was performed using an accelerated rotarod paradigm,wherein the latency to falling off the accelerating rod is a measure ofvestibulomotor function and motor coordination. MitoPark mice at 20weeks displayed significantly less time on the rotarod compared tocontrol mice (FIG. 13 ). However, EcN¹ _(L-DOPA) treatment significantlyincreased the time spent on the rotarod in MitoPark mice, but theEcN_(Vec) treatment did not (FIG. 13 ). Neither EcN¹ _(L-DOPA) orEcN_(Vec) had any effect on control mice (FIG. 13 ). This study suggeststhat EcN¹ _(L-DOPA) protects against the progressive loss ofvestibulomotor function and motor coordination in the MitoPark mousemodel of PD.

Collectively, our data support a strong colonization profile of newlydeveloped L-DOPA-producing E. coli Nissle bacteria (EcN¹ _(L-DOPA)) andits excellent pharmacokinetics and profile of plasma L-DOPA levels.Notably, EcN¹ _(L-DOPA) treatment alleviates Parkinsonianneurobehavioral symptoms in a preclinical animal model of PD.Furthermore, EcN¹ _(L-DOPA) reduces anxiety and depressive behaviors andenhances motivational behavior in both control and PD mice. Thus E. coliNissle 1917 L-DOPA-producing bacteria could be a viable therapeutic as anovel L-DOPA delivery mechanism with minimal side effects as well as anew anti-depressant and anxiolytic probiotic for treating PD and otherneuropsychiatric disorders.

Example 10: Sequences

HpaB gene sequence (SEQ ID NO: 1):

    1 atgaaaccag aagatttccg cgccagtacc caacgtccgt tcaccgggga agagtatctg   61 aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcact 121 actcatccgg catttcgtaa tgcggcagcg tctgttgccc aactgtacga cgcgctgcac 181 aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 241 cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 301 gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagctgct 361 ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 421 aactggtata cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 481 ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 541 gagactgacg ccgggattat cgttagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 601 actcactaca acatggttgg cttcggctcg gcacaagtaa tgggcgaaaa cccggacttc 661 gcgctgatgt tcgttgcgcc aatggatgct gatggcgtga aattaatctc ccgcgcctct 721 tatgagatgg tcgcgggtgc taccggctca ccgtatgact acccgctctc cagccgcttc 781 gatgagaatg atgcgattct ggtgatggat aacgtgctga tcccatggga aaacgtgctg 841 atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 901 tatccgctgc aagcctgtgt gcgcctggca gtgaaactcg acttcattac ggcactgctg 961 aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt1021 gaagtggtgg cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttctgaagcg1081 acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta1141 ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc1201 ctgatctacc tcccttccag tgcccgtgac ctgaacaatc cgcagatcga ccagtatctg1261 gcgaagtatg tgcgcggttc gaatggtatg gatcacgtcc agcgcatcaa gatcctcaaa1321 ctgatgtggg acgccattgg cagcgagttt ggtggtcgcc acgaactgta tgaaatcaac1381 tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aagctccggc1441 aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccagaacggc1501 tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa1561 taa

The HpaC gene sequence (SEQ ID NO: 2):

   1 atgcaattag atgaacaacg cctgcgcttt cgtgacgcga tggccagcct gtcggcagcg 61 gtaaatatta tcaccaccga gggcgacacc ggacaatgcg ggattacggc aacggctgtc121 tgctcggtca cggatacacc accgtcgctg atggtgtgca ttaacgccaa cagtgcgatg181 aacccggttt ttcagggcaa cggcaagttg tgcgtcaacg tcctcaacca tgagcaggaa241 ctgatggcac gccacttcgc gggcatgaca ggcatggcga tggaagagcg ttttagcctc301 tcatgctggc aaaaaggtcc gctggcgcag ccggtgctaa aaggttcgct ggccagtctt361 gaaggtgaga tccgcgatgt gcaggcaatt ggcacacatc tggtgtatct ggtggagatt421 aaaaacatca tcctcagtgc agaaggtcac ggacttatct actttaaacg ccgtttccat481 ccggtgatgc tggaaatgga agctgcgatt taa

Example 11: Re-Engineered EcN² _(L-DOPA) Probiotic

We have cloned the codon-optimized L-DOPA biosynthesis genes4-hydroxyphenylacetate 3-monooxygenase (HpaB) and its FAD reductase(HpaC) into a reconstructed, BBa_J23111 promoter-based vector and stablytransformed EcN to express the HpaB and HpaC enzymes. We chose EcN asthe delivery vehicle for continuous in situ production of L-DOPA (EcN²_(L-DOPA)) because this commensal strain has proven to be a safe andeffective probiotic supplement for therapeutic development in humans.This example demonstrates that: 1) the genetically reengineered,second-generation L-DOPA-producing E. coli Nissle 1917 probiotic strain(EcN² _(L-DOPA)) produces and releases L-DOPA in vitro withoutsupplementing L-tyrosine, and 2) oral administration of this highlyefficient EcN² _(L-) _(DOPA) strain readily colonizes the mouse gut,achieves a steady-state plasma L-DOPA level that corresponds to theclinically effective plasma level in PD patients, and increases L-DOPAand dopamine levels in the brain.

We confirmed via HPLC that our 2^(nd)-generation recombinant E. coliNissle 1917 strain expressing HpaBC genes (EcN² _(L-DOPA)) was moreefficient and effective than the 1^(st)-generation L-DOPA-expressingsystem (EcN¹ _(L-DOPA)) at producing large amounts of L-DOPA inbacterial culture media, even without supplementing with L-tyrosine(FIGS. 14 ). Importantly, we also demonstrated that the geneticallyre-engineered EcN² _(L-DOPA) colonized the gut of C57BL mice,constitutively produced stable levels of plasma L-DOPA corresponding toclinically effective plasma levels in PD patients, and significantlyincreased striatal L-DOPA and DA (FIGS. 15-17 ). We achieved statisticalsignificance using 4-6 mice/ group, indicating a robust L-DOPAreplacement strategy. Neurochemical and colonization assays were run inblinded triplicates. In addition, a pilot study using a relatively smallnumber of animals indicates that oral treatment with EcN² _(L-DOPA) onalternate days for 1 wk alleviates key motor deficits in MitoPark (MP)mice, a chronically progressive neurodegenerative mouse model of PD.

Characterization and Optimization of Microbial L-DOPA Synthesis inGenetically Engineered Probiotic E. coli.

We optimized the system’s translational versatility and flexibility byreconstructing the L-DOPA-expressing plasmid. We used strong syntheticconstitutive σ⁷⁰ promoters from the Registry of Standard BiologicalParts (parts.igem.org/Catalog) and optimized HpaBC gene codons forexpression in E. coli. Briefly, the native promoter of a pRham-Kanamycinvector was replaced with the constitutive synthetic promotersBBa_J23100, BBa_J23105, and BBa_J23111, herein referred as P1, P2 andP3, respectively, which are derived from the consensus promoterBBa_J23119 and have relative reported activities tested in the TG1strain as 1, 0.24, and 0.58 for P1, P2, and P3, respectively. AnIDT-synthesized gBlocks gene fragment containing codon-optimized HpaBCgenes was inserted into the generated P1, P2 and P3 vectors (FIG. 14A).L-DOPA production from the EcN containing the newly constructedL-DOPA-producing plasmids was quantitatively assayed by HPLC.Surprisingly, even in LB media (normal L-tyrosine 1-1.4 mM) lackingL-tyrosine supplementation, all new systems were able to produce muchhigher concentrations of L-DOPA than the previous RSF1030-based EcN¹_(L-DOPA) system, resulting in a more gradual L-DOPA production withyield strengths for the P2-, P1-, and P3-based system being 5, 7, and 31times that of the RSF1030 system (FIG. 14B). Collectively, ouroptimization strategy has the advantage of allowing more effective andefficient L-DOPA production, even without adding L-tyrosine. Since ouroptimized P1-P3 EcN L-DOPA system synthesizes a graded level of L-DOPAwith a normal L-tyrosine level, this improved system allows us topersonalize L-DOPA delivery depending on each patient’s DA needs and tofine-tune dose titrations. Since our P3-based L-DOPA-expressing system(referred to as EcN² _(L-DOPA)) yielded the highest L-DOPA production(FIG. 14B), we used it for the following in vivo preliminary studies.

EcN² _(L-DOPA) Colonization and L-DOPA Production in a Mouse Model

We used C57BL/6NCrl mice to test whether orally administered EcN²_(L-DOPA) colonizes the mouse gut and effectively elevates plasmaL-DOPA. For colonization studies, fecal pellets were collected dailyfrom mice for 4 days following a single oral dose of 10⁹ CFU (colonyforming unit) P3-based EcN² _(L-D)o_(PA) or PBS and the peripheral AADCinhibitor benserazide (Bz, 12.5 mg/kg, i.p.) to prevent breakdown ofL-DOPA. EcN² _(L-DOPA) colonization of the mouse gut increased rapidly(FIG. 15A), peaking at days 1-2 post-challenge (4.8 ×10⁷ cfu/g), andthen gradually declined until it dropped to baseline on days 3-4, asrevealed by qPCR detection of a unique sequence for the EcN strainpresent in the fecal pellets following a previous method. Thecolonization profile suggests that EcN² _(L-) _(DOPA) treatment onalternate days represents a suitable paradigm for achieving stable EcN²_(L-) _(DOPA) gut colonization in mice. Next, we examined whether EcN²_(L-D)o_(PA) treatment on alternate days leads to stable plasma L-DOPAlevels. Mice were orally administered a single daily dose of 10⁹ CFUEcN² _(L-DOPA) or PBS and also Bz (50 mg/kg) 3 times/day for 1 and 2days. Plasma samples collected through submandibular sampling prior toEcN² _(L-DOPA) treatment (serving as baseline), and at 4, 8, 16, 24 and48 h post-EcN² _(L-DOPA) and Bz (50 mg/kg) treatment, were assayed forL-DOPA production by HPLC. We found that following EcN² _(L-D)o_(PA)administration, plasma levels of L-DOPA gradually increased over time,reaching 1387.6 ng/ml at 1 day and then remaining relatively stableuntil 2 days post-treatment (FIG. 15B). We increased the dose andfrequency of Bz because of its short half-life. This repeated Bztreatment paradigm prolonged Bz plasma levels up to 30 h as determinedby HPLC (FIG. 16A). These results are clinically important since theoptimal therapeutic plasma levels of L-DOPA are considered to be in therange of 300-1600 ng/ml in humans. The plasma L-DOPA profiles suggestthat therapeutic plasma levels of L-DOPA can be achieved and maintainedstably for at least 48 h following oral administration of a single doseof EcN² _(L-DOPA). Given these results, combined with the colonizationprofile showing highest colonization at 1-2 days post EcN² _(L-DOPA)gavage, we believe that treatment with a single dose of EcN² _(L-DOPA)in mice on alternate days will induce stable colonization and continuousL-DOPA delivery. Analysis of striatal L-DOPA levels revealed that asingle dose of 10⁹ CFU EcN² _(L-DOPA) and repeated Bz (50 mg/kg)treatment significantly increased L-DOPA levels compared to PBS controlmice (FIG. 16B). Importantly, DA levels in striatal tissues were alsodramatically increased in mice orally administered with EcN² _(L-DOPA)for 1-2 days as compared to control animals (FIG. 16C, 2.9- and 2.5-foldincrease for 24 h and 48 h post-EcN² _(L-DOPA), respectively). EcN²_(L-) _(DOPA) colonization at 48 h post-EcN² _(L-DOPA) was alsodetermined by qPCR detection of EcN’s unique sequence after isolatingtotal DNAs from the intestinal content collected from region-specificgut segments, including duodenum, ileum, cecum and colon. The intestinalcontent collected from all gut segments showed significant levels of EcNbacteria (FIG. 16D), suggesting our genetically reengineered bacteriumsuccessfully colonized throughout the mouse gut.

To further evaluate whether EcN_(L-DOPA) adapts well in the gut withoutproducing any significant adverse effect in the target tissue,histopathological and clinical chemistry analyses were performed. Wetreated animals with a single dose (10⁹ CFU) of the 1^(st)-generationsystem (EcN¹ _(L-DOPA)) or PBS for 7 days. Histological analyses of theileum, cecum and colon of EcN¹ _(L-DOPA)-treated animals and blindedevaluation of histological scores for inflammation, edema, stomalcollapse, gland hyperplasia, and distribution revealed no pathologicalchanges (FIG. 17A), suggesting that EcN¹ _(L-DOPA) treatment did notinduce toxicity, inflammation, or neoplastic processes in the mouse gut.Blood chemistry results consistently showed no significant differencebetween control and EcN¹ _(L-DOPA)-treated mice (Table 1). In addition,to further determine if EcN¹ _(L-DOPA) induces any dysbiosis orimbalance in the gut microbiota, we performed fecal metagenomicanalyses. No significant differences in the key family of bacteria wereobserved, indicating that EcN¹ _(L-DOPA) does not negatively impact gutmicrobiota (FIG. 17B). Together, these results suggest that colonizedEcN² _(L-DOPA) efficiently produce stable levels of L-DOPA in micecorresponding to clinically effective plasma levels in human subjectswithout any significant adverse effect, and the microbially deliveredL-DOPA crosses the BBB to the brain where it is further converted toenhance levels of DA.

EcN² _(L-DOPA) Treatment Alleviates Locomotor Deficits in MP Mice

To examine the effects of EcN² _(L-DOPA) on locomotor deficits, weperformed a pilot study using a small number of 11- to 16-wk-old MP andlittermate control mice (n=4-5/group), which were gender-balanced andblindly randomized to groups orally administered a single dose of either10⁹ CFU EcN² _(L-DOPA) or PBS in combination with Bz on alternate daysfor 1 wk. All mice were assessed post-treatment for open-field locomotoractivity using the VersaMax computerized activity monitoring system.Representative maps of movement paths (FIG. 18A) and analyses of meanhorizontal activity (FIG. 18B) revealed that 12- to 17-wk MP micedisplayed significantly reduced locomotor activity compared tolittermate control mice and that orally administered EcN² _(L-DOPA)increased locomotor activity in MP mice. Moreover, MP mice exhibiteddecreased stereotypy (FIG. 18C) compared to control mice. Importantly,orally administered EcN² _(L-DOPA) significantly attenuated motordeficits in MP mice while having no significant effect in control mice.Together, these results clearly suggest that alternate-day treatmentswith EcN² _(L-DOPA) for 1 wk alleviate key motor deficits in the MPmouse model of PD.

Please note that we used a PBS control only in early experiments, but wehave now generated EcN vector control (EcN_(Vec)) data showing nosignificant change in plasma L-DOPA levels compared to PBS controls(FIG. 19 ).

The newly constructed 2^(nd)-generation L-DOPA-expressing systems allowa gradual and more efficient L-DOPA production without supplementing theL-tyrosine substrate, providing more flexibility in terms of dosetitration than the 1st-generation (RSF1030-based) system. We have strongprelim data demonstrating our 2^(nd)-generation EcN² _(L-DOPA) colonizesthe mouse gut, produces stable therapeutic plasma levels of L-DOPA andincreases brain DA levels. As an alternate approach, we will considerusing inducible control of L-DOPA synthesis as a strategy to achievebetter therapeutic efficacy in relieving PD symptoms and preventing LIDdevelopment. An inducible control of L-DOPA production is also valuablein preventing overmedication and related harmful drug reactions.Although EcN² _(L-DOPA) has a kanamycin-resistant plasmid, high doses ofantibiotics can eliminate the EcN² _(L-DOPA) bacteria. The combinationof antibiotic treatment and transient colonization over 2-3 days offeran adequate mitigation strategy in case of any overmedication or adversedrug reaction with EcN² _(L-DOPA) treatment. To further improve theversatility and flexibility of our L-DOPA-expressing systems, we willconsider developing a chromosomal integration and expression system forL-DOPA production.

We recognize that the clinical utility of EcN² _(L-DOPA) treatment in PDpatients will require further optimization depending on the gut motilityrate, dietary factor and severity of the disease. Since our optimizedP1-P3 EcN L-DOPA system (FIGS. 14 ) synthesizes a graded level of L-DOPAwith normal L-tyrosine level, this improved system can be adopted topersonalize the L-DOPA delivery depending on each patient’s DA needs andto carry out up- and down-titration of L-DOPA medication in clinicalsettings. The plasma L-DOPA level can be used as an index for up/downtitration to optimize the correct dose needed for patients.

hpaBC Cloning and Expression

To create an E. coli Nissle (EcN) strain to produce elevated levels ofL-DOPA we first assembled recombinant plasmids to express a synthetichpaBC gene construct. The hpaBC genes collectively encode4-hydroxyphenylacetate-3-hydrolase, which converts L-Tyrosine to L-DOPAand are found naturally in selected E. coli strains. A codon-optimizedvariant was synthesized (Integrated DNA Technologies, Coralville IA).This new hpaBC variant shared 79% nucleotide sequence identity with theoriginal genes and included additional bases on the 5′ and 3′ ends tofacilitate cloning (SEQ ID NO: 3).

The synthetic hpaBC genes were cloned under control of the rhamanose(rha) promoter and operator for expression in EcN. For this, thecommercially available pRHAM vector (Lucigen, Madison WI) was used.Kanamycin resistant transformants were screened for synthesis of L-Dopain the presence of 1% rhamnose for dark colonies, indicative ofoxidation of L-DOPA to dopachrome with subsequent polymerization to formthe pigment melanin (Claus, H., and H. Decker. 2006. Bacterialtyrosinases. Syst. Appl. Microbiol. 29:3-14). Correct insertion of hpaBCinto the pRham vector (vector pRham-hpaBC_(syn)) was confirmed bycharacterizing plasmid DNA by restriction enzyme digestion and DNAsequencing.

The pRham-hpaBC_(syn) plasmid was introduced to EcN by chemicaltransformation for characterization in the experiments describedelsewhere. Additional variants of pRham-hpaBC_(syn) were also made toreplace the rha promoter with promoters that allow constitutiveexpression of hpaBC. Three synthetic σ⁷⁰ promoter sequences (“parts”BBa_J23100 [P1], BBa_J23105 [P2], and BBa_J23111 [P3]) from the AndersonPromoter Collection (iGEM.org) were selected based on different levelsof transcriptional activity afforded by each sequence. Each promotersequences were incorporated into PCR primers used for inverse PCRreactions using pRham-hpaBC_(syn) as template DNA. Transformantsconstitutively expressing rhaBC were identified by colonies that turneddark brown following incubation in the absence of L-rhamnose. Aspredicted, expression of hpaBC from promoters P1, P2, and P3 (FIG. 20 )resulted in different levels of constitutive L-DOPA production.

hpaBC_(syn) nucleotide sequence (SEQ ID NO: 3):

GAAGGAGATATACATATGAAACCCGAAGATTTCCGTGCTTCAACACAGCGCCCTTTCACTGGGGAAGAATACctgAAGAGCCTGCAAGACGGTCGTGAAATTTATATTTACGGGGAGCGTGTGAAGGATGTTACGACCCATCCAGCCTTTCGCAACGCCGCTGCGTCTGTGGCGCAGTTGTATGATGCGTTACACAAACCTGAGATGCAGGATTCGTTGTGCTGGAACACAGACACGGGTTCGGGAGGATATACTCATAAATTTTTTCGCGTTGCTAAGTCGGCAGACGACCTgCGCCAACAACGTGATGCTATTGCTGAGTGGTCACGTCTGTCGTACGGGTGGATGGGACGTACACCCGATTATAAAGCGGCGTTTGGATGCGCATTGGGAGCTAACCCTGGATTCTATGGACAGTTCGAGCAGAATGCCCGCAACTGGTACACACGCATTCAAGAAACTGGGTTGTATTTTAATCACGCCATTGTCAATCCGCCGATCGATCGCCACCTGCCCACGGATAAAGTAAAAGATGTATATATTAAGTTGGAAAAAGAGACAGACGCAGGGATCATTGTATCAGGCGCCAAGGTGGTTGCGACCAATTCTGCCCTGACGCACTACAACATGATCGGCTTTGGATCTGCTCAAGTGATGGGTGAAAACCCCGATTTTGCACTTATGTTTGTAGCCCCCATGGACGCTGACGGGGTTAAACTGATTAGCCGCGCATCGTACGAAATGGTCGCCGGGGCCACAGGCAGTCCGTACGATTATCCTTTATCTAGTCGCTTCGACGAAAACGACGCGATCTTAGTGATGGATAACGTCCTGATTCCTTGGGAGAACGTCCTGATCTATCGTGATTTCGACCGCTGCCGTCGTTGGACTATGGAAGGAGGCTTCGCTCGCATGTACCCTTTGCAAGCCTGTGTACGCCTTGCTGTCAAACTTGATTTCATCACTGCGCTTTTGAAGAAATCGTTAGAGTGTACTGGGACGCTGGAGTTCCGTGGTGTCCAAGCCGACCTTGGCGAGGTGGTGGCTTGGCGTAATACTTTCTGGGCATTATCCGACTCCATGTGCTCGGAAGCAACCCCCTGGGTCAATGGGGCATACCTTCCCGATCACGCCGCTCTTCAAACCTATCGCGTACTTGCGCCTATGGCtTATGCTAAGATTAAAAATATTATCGAACGTAATGTGAGTTCCGGCTTAATTTACTTGCCCTCCAGCGCGCGCGATCTGAATAATCCTCAAATCGACCAGTATTTAGCGAAGTATGTTCGCGGGAGCAACGGGATGGATCATGTCCAGCGCATCAAAATCCTTAAGTTAATGTGGGATGCCATTGGTTCAGAATTTGGCGGGCGTCATGAACTTTATGAAATTAATTACTCTGGCTCGCAAGACGAGATCCGTctgCAATGCTTGCGCCAGGCGCAATCCTCGGGTAATATGGATAAGATGATGGCTATGGTAGACCGCTGCCTGTCCGAGTACGACCAAAACGGATGGACGGTCCCCCATCTGCATAACAACGATGACATTAACATGCTGGATAAGctgctgAAATAACGCAGCAGGAGGTTAAGATGCAGcTGGACGAACAACGCctgCGCTTTCGTGACGCAATGGCGAGTTTGAGTGCAGCCGTTAATATCATTACAACAGAAGGGGACGCAGGTCAATGTGGAATCACTGCAACGGCCGTGTGCTCAGTTACAGACACTCCGCCTTCATTAATGGTATGCATCAATGCTAACTCGGCTATGAATCCTGTCTTCCAAGGCAATGGGAAATTATGTGTGAACGTCCTGAACCATGAGCAAGAAcTGATGGCACGCCACTTCGCAGGCATGACAGGTATGGCAATGGAGGAGCGTTTTAGCTTGTCTTGCTGGCAAAAGGGACCActgGCCCAACCAGTTTTGAAGGGGTCTCTTGCATCATTAGAAGGGGAAATCCGCGATGTCCAGGCGATTGGTACACACCTgGTTTACCTTGTCGAGATCAAGAACATCATTTTATCCGCAGAGGGCCACGGGCTGATTTACTTTAAACGCCGCTTCCATCCGGTTATGCTGGAAATGGAAGCTGCAATTTAAGTAAGGAAACATTTATGCGCCTGCATCATCACCACCATCAC 

BBa_J23100 TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC (SE Q ID NO: 4) 

BBa_J23105 TTTACGGCTAGCTCAGTCCTAGGTACTATGCTAGC (SE Q ID NO: 5) 

BBa_J23111 TTGACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC (SE Q ID NO: 6)

Example 12: Re-Engineered L-DOPA Microbiome Therapy for Depression andAnxiety

Major depressive disorder (MDD) is a recurrent or chronic mood disordercharacterized by experiencing a depressed mood or loss of interest thatis present across most situations for at least two weeks. This disorderresults in severe psychosocial dysfunction that commonly leads tofeelings of hopelessness, helplessness, anxiety and negativity, thusaffecting a person’s daily activities and general health. Severedepression may lead some people to feel as if life isn’t worth living.As one of the most common mental disorder in the world, almost one infive people experiences one episode at some point in their lifetime andover 350 million people of all ages around the world and 17 millionAmerican adults are estimated to suffer this disease. As exposure tohighly stressful life experiences is one of the most consistentlydocumented risk factors for depression, deployment to combat zones andexposure to combat have long been associated with increased risk ofdepression and other mental health problems. Several lines of evidenceindicate that depression is as common as, or perhaps even more commonthan post-traumatic stress disorder (PTSD) among combat veterans.Epidemiological studies estimated the prevalence of depression amongU.S. military personnel to be 12% among currently deployed, 13.1% amongpreviously deployed, and 5.7% among never deployed. Due to its variouspresentations, unpredictable course and prognosis, and variable responseto treatment, depression often pose huge challenges for clinicians.

The pathophysiology of depression is not yet known, but considerableevidence supports that insufficient activity of monoamineneurotransmitters, increased inflammation, andhypothalamic-pituitary-adrenal (HPA) axis abnormalities contribute todepression. Depression has been shown to integrate circuitry fromseveral neuronal pathways including the noradrenergic, dopaminergic andserotonergic for which it is likely that each unique circuit is involvedin the presentation of its symptoms. Traditionally, dysfunction of thehighly complex integrated noradrenergic and serotonergic circuitry wasassociated with the disorder. However, with recent development inneuroimaging and electrophysiological technology, depression modelled inchronic stress rodents indicated that damage within the mesolimbic andcorticolimbic dopaminergic circuits leads to MDD symptoms (loss ofmotivation arousal, impairment in reward prediction, and anhedonia).Reduced concentrations of dopamine metabolites have been detected inpostmortem investigation. In mesolimbic pathway, dopaminergic neuronsoriginate from the ventral tegmental area (VTA) and project its axon tonucleus accumbens (NAc). In animal models of depression, depletion of DAfrom VTA dopaminergic neurons showed an altered activation of its limbictargets. Optogenetic stimulation of VTA resulted in a reduction ofbehavioral deficits and dopaminergic neuronal cell firing. These effectswere reversed when blocking with dopamine receptor antagonists in theNAc. In the corticolimbic pathway, the VTA primarily projects to theprefrontal cortex (PFC) and the anterior cingulate cortex (ACC). Studiesin stress-induced rat models have shown that PFC DA neurotransmissionexerts an inhibitory control of DA activity in NAc during stress. Thecorticolimbic DA system dulls DA release in the mesolimbic pathwaysleading to maintenance of depression-like behavior, for which effectswere reversed with depletion of DA in PFC and the anti-depressanttherapy. Similarly, the neuroendocrine system is highly influenced bythe integrated circuit of the noradrenergic and dopaminergic pathway. Inchronic stress rodents, activation of the HPA-axis triggers the releaseof glucocorticoids, corticotrophin releasing hormones (CRH) andpro-inflammatory cytokines (TNF, IL-1, IL-6). It was proven that understress and depression, DA, NE and 5-HT transmission is disrupted leadingto imbalance and impairment of regulatory feed-back loop responsible forturning off the stress response.

The antidepressants selective serotonin reuptake inhibitors (SSRI)followed by the noradrenaline reuptake inhibitors (NRI) that boostserotonin and/or noradrenaline neurotransmission, respectively, are themost commonly medications prescribed. However, both groups of drugs taketime- usually four to eight weeks- to work, and show efficacy in only60-70% of patients. Several strategies have been developed to increasethe efficacy of the SSRI and NRI, including using concomitantly anantidepressant with a substance from another drug class, such as thoseacting on the dopaminergic system. Growing evidence supports the use ofdopamine receptor agonist as antidepressant. Currently, manipulation ofdopamine contractions in the brain is primarily achieved through thedopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) treatment.L-DOPA readily enters the brain, where it is converted into dopamine bythe enzyme DOPA decarboxylase (DDC). Evidence has suggested thatco-administration with L-DOPA enhances the anti-depressive effects ofNRIs. Furthermore, the L-DOPA-converted dopamine can be furtherconverted to noradrenaline by the enzyme dopamine β-monooxygenase, andL-DOPA itself can also be stored by serotonergic afferents, suggestingthat L-DOPA acts in the context of depression via various mechanisms.Regrettably, the use of orally tablet dosing of L-DOPA over time resultsin diminished production of endogenous L-DOPA. More importantly, due toits chronically non-continuous delivery of L-DOPA to the brain,long-term conventional L-DOPA treatment often induces an array of motorfluctuations and other severe complications including dyskinesia. Wedemonstrated that the EcN_(L-DOPA) colonized the gut of C57BL mice, andconstitutively produced plasma L-DOPA. Importantly, the neurotransmitterlevels of NE, 5-HT and DA in the frontal cortex dramatically increasedfor 2-16 h in mice orally administered with a single dose of 10⁹ CFUEcN_(L-DOPA) and Bz (FIGS. 21A-C). Surprisingly, administration ofEcN_(L-DOPA) improved depression- and anxiety-like behavior in controlC57BL mice (FIGS. 22 ).

Antidepressant and Anxiolytic Effects of EcN_(L-DOPA) in Mice

Depression and anxiety were assessed in mice using the forced swim test(FST), open-field test (OFT) and elevated plus maze (EPM). FST subjectsmice to an inescapable, stress-inducing task that invariably leads tobehavioral despair as measured by lower mobility. The OFT relies on amouse’s natural tendency to hug opaque walls, avoiding openly exposedareas when spontaneously exploring novel areas. More time spent in themost openly exposed areas serves as an index of reduced anxiety. The EPMmeasures anxiety based on the mouse’s positive thigmotactic aversion toopen spaces, with reduced anxiety indicated by more time spent in theopen arms and higher total distance traveled. C57BL/6NCrl mice orallyreceiving a single dose of 10⁹ CFU EcN_(L-DOPA) and 12.5 mg/kg Bz onalternate days for one month were significantly less anxious than or PBSvehicle-treated control mice as evidenced by the representative heat mapshowing more time spent in the most openly exposed OFT quadrant (Q2)relative to the least exposed, opposite corner and increased overallhorizontal activity (FIG. 22A) and by increased time spent in open armsand total distances in EPM (FIG. 22B). When compared to control mice,mice receiving EcN_(L-DOPA) were significantly more mobile andcumulatively moved a significantly greater distance in the FST (FIG.22C), suggesting the mice were highly motivated to escape rather thanjust tread water, indicating that EcN_(L-DOPA) effectively treatsdepression-like motivational impairments in otherwise healthyindividuals. Our behavior results clearly demonstrate the antidepressantand anxiolytic effects of EcN_(L-DOPA) in a mouse model.

1-29. (canceled)
 30. A pharmaceutical composition comprising: arecombinant microbial cell capable of producing L-DOPA and colonizingthe gut of a subject, thereby providing L-DOPA in a sustained manner,and a carrier.
 31. The pharmaceutical composition of claim 30, whereinsaid recombinant microbial cell is a probiotic.
 32. The pharmaceuticalcomposition of claim 31, wherein said probiotic is E. coli Nissle 1917.33. The pharmaceutical composition of claim 30, wherein said compositioncomprises from about 10⁶ CFU to about 10¹² CFU of said recombinantmicrobial cell.
 34. The pharmaceutical composition of claim 33, whereinsaid composition comprises about 10⁹ CFU of said recombinant microbialcell.
 35. The pharmaceutical composition of claim 30, wherein saidcomposition further comprises L-Tyrosine.
 36. The pharmaceuticalcomposition of claim 30, wherein said recombinant microbial cellcomprises a hpaB and hpaC nucleotide sequence as set forth in SEQ ID NO:1 and 2 or as set forth in SEQ ID NO:
 3. 37. The pharmaceuticalcomposition of claim 36, wherein said hpaB and hpaC nucleotide sequenceis operably linked to promoter sequence comprising one or more of SEQ IDNOs: 4-6.
 38. (canceled)
 39. The pharmaceutical composition of claim 30for use in the treatment of Parkinson’s disease and other Parkinsonismsyndromes.
 40. The pharmaceutical composition of claim 30 for use in thetreatment of depression and/or anxiety.
 41. A recombinant microbial cellcapable of producing L-DOPA and colonizing the gut of a subject, therebyproviding L-DOPA in a sustained manner.
 42. The recombinant microbialcell of claim 41, wherein said recombinant microbial cell is aprobiotic.
 43. The recombinant microbial cell of claim 42, wherein saidprobiotic is E. coli Nissle
 1917. 44. The recombinant microbial cell ofclaim 41, wherein said composition comprises from about 10⁶ CFU to about10¹² CFU of said recombinant microbial cell.
 45. The recombinantmicrobial cell of claim 44, wherein said composition comprises about 10⁹CFU of said recombinant microbial cell.
 46. The recombinant microbialcell of claim 41, wherein said composition further comprises L-Tyrosine.47. The recombinant microbial cell of claim 41, wherein said recombinantmicrobial cell comprises a hpaB and hpaC nucleotide sequence as setforth in SEQ ID NO: 1 and 2 or as set forth in SEQ ID NO:
 3. 48. Therecombinant microbial cell of claim 47, wherein said hpaB and hpaCnucleotide sequence is operably linked to promoter sequence comprisingone or more of SEQ ID NOs: 4-6.
 49. The recombinant microbial cell ofclaim 41 for use in the treatment of Parkinson’s disease and otherParkinsonism syndromes.
 50. The recombinant microbial cell of claim 41for use in the treatment of depression and/or anxiety.
 51. A nucleotidesequence encoding hpaB and hpaC operably linked to a heterologouspromoter sequence and comprising one or more of SEQ ID NOs: 4-6.